Combination therapy with anti-HLA-DR antibodies and kinase inhibitors in hematopoietic cancers

ABSTRACT

The present invention relates to combination therapy with drugs, such as Bruton&#39;s tyrosine kinase inhibitors or PI3K inhibitors, with antibodies or ADCs against HLA-DR. Where ADCs are used, they preferably incorporate SN-38 or pro-2PDOX. The ADC may be administered at a dosage of between 1 mg/kg and 18 mg/kg, preferably 4, 6, 8, 9, 10, 12, 16 or 18 mg/kg. The combination therapy can reduce solid tumors in size, reduce or eliminate metastases and is effective to treat cancers resistant to standard therapies, such as radiation therapy, chemotherapy or immunotherapy. Preferably, the combination therapy has an additive effect on inhibiting tumor growth. Most preferably, the combination therapy has a synergistic effect on inhibiting tumor growth.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/484,308, filed Apr. 11, 2017, which was a continuation-in-part ofU.S. patent application Ser. No. 15/190,805 (now issued U.S. Pat. No.9,707,302), filed Jun. 23, 2016, which claimed the benefit under 35U.S.C. 119(e) of U.S. Provisional Patent Application Nos. 62/184,331,filed Jun. 25, 2015, 62/201,361, filed Aug. 5, 2015, 62/250,715, filedNov. 4, 2015 and 62/263,134, filed Dec. 4, 2015. U.S. patent applicationSer. No. 15/484,308 claimed the benefit under 35 U.S.C. 119(e) of U.S.Provisional Patent Application No. 62/322,441, filed Apr. 14, 2016, and62/373,591, filed Aug. 11, 2016. This application is a divisional ofU.S. patent application Ser. No. 15/353,141, filed Nov. 16, 2016, whichwas a divisional of U.S. patent application Ser. No. 14/878,715 (nowabandoned), filed Oct. 8, 2015, which was a divisional of U.S. patentapplication Ser. No. 14/224,866 (now issued U.S. Pat. No. 9,187,561),filed Mar. 25, 2014, which was a divisional of U.S. patent applicationSer. No. 12/754,140 (now issued U.S. Pat. No. 8,722,047), filed Apr. 5,2010, which was a continuation-in-part of U.S. patent application Ser.No. 12/556,718 (now issued U.S. Pat. No. 8,613,903), filed Oct. 10,2009, which was a divisional of U.S. patent application Ser. No.11/368,296 (now issued U.S. Pat. No. 7,612,180), filed Mar. 3, 2006,which claimed the benefit under 35 USC 119(e) of provisional U.S. PatentApplication Ser. No. 60/657,695 filed on Mar. 3, 2005. U.S. patentapplication Ser. No. 12/754,140 (now issued U.S. Pat. No. 8,722,047),claimed the benefit under 35 U.S.C. 119(e) of U.S. Provisional PatentApplication Ser. Nos. 61/166,809, filed Apr. 6, 2009, and 61/168,715,filed Apr. 13, 2009. The entire text of each priority application isincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 27, 2017, isnamed IMM367US1_SL.txt and is 7,866 bytes in size.

FIELD OF THE INVENTION

The present invention relates to therapeutic use in hematopoietic cancerof anti-HLA-DR antibodies or antibody-drug conjugates (ADCs), incombination with one or more kinase inhibitors, wherein the combinationtherapy is more effective than the antibody or ADC alone, kinaseinhibitor alone, or the combined effects of kinase inhibitor andantibody or ADC alone. In preferred embodiments, the combinationexhibits synergistic effects. In other preferred embodiments, the kinaseinhibitors of use are Bruton's kinase inhibitors or phosphoinositide3-kinase (PI3K) inhibitors. The kinase inhibitors may be administeredseparately or together with the antibodies, or may be conjugated to theantibody prior to administration. In the latter case, the antibodies andkinase inhibitors may be linked via an intracellularly-cleavable linkagethat increases therapeutic efficacy. In alternative embodiments, theanti-HLA-DR antibody may be conjugated to a different drug (such asSN-38) to form an ADC, and the ADC may be administered in combinationwith a Bruton's kinase inhibitor or PI3K inhibitor. Preferably, ADCs areadministered at specific dosages and/or specific schedules ofadministration that optimize their therapeutic effect. The optimizeddosages and schedules of administration of ADCs for human therapeuticuse disclosed herein show unexpected superior efficacy that could nothave been predicted from animal model studies, allowing effectivetreatment of cancers that are resistant to standard anti-cancertherapies, including irinotecan (CPT-11), paclitaxel or other compounds.In a particularly preferred embodiment, an anti-HLA-DR antibody of useis IMMU-114 (hL243).

BACKGROUND OF THE INVENTION

Rituximab anti-CD20 IgG therapy is credited with revitalizing antibodytherapies with its ability to effectively treat follicular lymphomawithout the extensive side effects associated with more traditionalchemotherapy regimens. Since rituximab's approval by the FDA in 1997,the mortality rate from NHL has declined by 2.8% per year (Molina, 2008,Ann Rev Med 59:237-50), and the use of this agent has been expanded to avariety of diseases. While rituximab has been a remarkable success infollicular non-Hodgkin lymphoma (NHL), for which it was first approved,only half of the patients had an objective response, with at most 10%having a complete response (McLaughlin et al., 1998, J Clin Oncol16:2825-33). Rituximab was less effective in the more aggressive typesof NHL, such as diffuse large B cell lymphoma (DLBCL), but when it wascombined with combination chemotherapy, improved and durable objectiveresponses compared to the separate therapies were found, making R-CHOP astandard protocol for the treatment of DLBCL (e.g., Leonard et al.,2008, Semin Hematol 45:S11-16; Friedberg et al., 2002, Br J Haematol117:828-34). The success of rituximab stimulated the evaluation of anumber of other antibodies and antibody conjugates, and while a numberof these have shown promising activity, to-date only one otherunconjugated antibody therapy, alemtuzumab anti-CD52 for chroniclymphocytic leukemia (CLL), has been approved for use in hematologicmalignancies (Robak, 2008, Curr Cancer Drug Targets 8:156-71).

The human leukocyte antigen-DR (HLA-DR) is one of three isotypes of themajor histocompatibilty complex (MHC) class II antigens. HLA-DR ishighly expressed on a variety of hematologic malignancies and has beenactively pursued for antibody-based lymphoma therapy (Brown et al.,2001, Clin Lymphoma 2:188-90; DeNardo et al., 2005, Clin Cancer Res11:7075s-9s; Stein et al., 2006, Blood 108:2736-44). Preliminary studiesindicate that anti-HLA-DR mAbs are markedly more potent than other nakedmAbs of current clinical interest in in vitro and in vivo experiments inlymphomas, leukemias, and multiple myeloma (Stein et al., unpublishedresults). HLA-DR is also expressed on a subset of normal immune cells,including B cells, monocytes/macrophages, Langerhans cells, dendriticcells, and activated T cells (Dechant et al., 2003, Semin Oncol30:465-75). Thus, it is perhaps not surprising that infusionaltoxicities, likely related to complement activation, have beenproblematic clinically with the administration of anti-HLA-DR antibody(Shi et al., 2002, Leuk Lymphoma 43:1303-12.

A need exists for improved compositions and methods of administration ofanti-HLA-DR antibodies or ADCs, alone or in combination with othertherapeutic agents, such as kinase inhibitors, for better therapeuticefficacy and decreased systemic toxicity in the treatment ofhematopoietic cancers, such as acute lymphoblastic leukemia (ALL) andchronic lymphocytic leukemia (CLL).

SUMMARY OF THE INVENTION

In preferred embodiments, the invention involves combination therapyusing an anti-HLA-DR antibody, or ADCs thereof, in combination with akinase inhibitor selected from the group consisting of Bruton's kinaseinhibitors and phosphoinositide 3-kinase (PI3K) inhibitors. Thecombination therapy is more effective than antibody or ADC alone, kinaseinhibitor alone, or the sum of the effects of antibody or ADC and kinaseinhibitor. Most preferably, the combination exhibits synergistic effectsfor treatment of hematopoietic cancer in human subjects. In embodimentsinvolving use of ADCs, the antibody is preferably conjugated to a CPTmoiety, such as SN-38, or to an anthracycline, such as pro-2PDOX. Asused herein, the abbreviation “CPT” may refer to camptothecin or any ofits derivatives, unless expressly stated otherwise. Preferably, thecamptothecin is SN-38.

The anti-HLA-DR antibody can be of various isotypes, preferably humanIgG1, IgG2, IgG3 or IgG4, more preferably comprising human IgG1 hingeand constant region sequences. The antibody or fragment thereof can be achimeric human-mouse, a chimeric human-primate, a humanized (humanframework and murine hypervariable (CDR) regions), or fully humanantibody, as well as variations thereof, such as half-IgG4 antibodies(referred to as “unibodies”), as described by van der Neut Kolfschotenet al. (Science 2007; 317:1554-1557). More preferably, the antibody orfragment thereof may be designed or selected to comprise human constantregion sequences that belong to specific allotypes, which may result inreduced immunogenicity when the antibody or ADC is administered to ahuman subject. Preferred allotypes for administration include a non-G1m1allotype (nG1m1), such as G1m3, G1m3,1, G1m3,2 or G1m3,1,2. Morepreferably, the allotype is selected from the group consisting of thenG1m1, G1m3, nG1m1, 2 and Km3 allotypes.

In alternative embodiments, the antibody of use may bind to atumor-associated antigen (TAA) other than HLA-DR. Many TAAs are known inthe art, including but not limited to, carbonic anhydrase IX,alpha-fetoprotein (AFP), α-actinin-4, A3, antigen specific for A33antibody, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1,CASP-8/m, CCL19, CCL21, CD1, CD1a, CD2, CD3, CD4, CD S, CD8, CD11A,CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30,CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54,CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83,CD95, CD126, CD132, CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A,CTLA-4, CXCR4, CXCR7, CXCL12, HIF-1α, colon-specific antigen-p (CSAp),CEA (CEACAM-5), CEACAM-6, c-Met, DAM, EGFR, EGFRvIII, EGP-1 (Trop-2),EGP-2, ELF2-M, Ep-CAM, fibroblast growth factor (FGF), Flt-1, Flt-3,folate receptor, G250 antigen, GAGE, gp100, GRO-β, HLA-DR, HM1.24, humanchorionic gonadotropin (HCG) and its subunits, HER2/neu, HMGB-1, hypoxiainducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α,IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6,IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growthfactor-1 (IGF-1), IGF-1R, KS1-4, Le-Y, LDR/FUT, macrophage migrationinhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3,mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13,MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin,PD-1 receptor, PD-Ll receptor, placental growth factor, p53, PLAGL2,prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF, ILGF, ILGF-1R, IL-6,IL-25, RS5, RANTES, T101, SAGE, 5100, survivin, survivin-2B, TAC,TAG-72, tenascin, TRAIL receptors, TNF-α, Tn antigen,Thomson-Friedenreich antigens, tumor necrosis antigens, VEGFR, ED-Bfibronectin, WT-1, 17-1A-antigen, complement factors C3, C3a, C3b, C5a,C5, an angiogenesis marker, bcl-2, bcl-6, Kras, an oncogene marker andan oncogene product (see, e.g., Sensi et al., Clin Cancer Res 2006,12:5023-32; Parmiani et al., J Immunol 2007, 178:1975-79; Novellino etal. Cancer Immunol Immunother 2005, 54:187-207). Preferably, theantibody binds to CEACAM-5, CEACAM-6, EGP-1 (Trop-2), MUC-16, AFP,MUC5a,c, CD74, CD19, CD20, CD22 or HLA-DR.

Exemplary antibodies that may be utilized in such alternativeembodiments include, but are not limited to, hR1 (anti-IGF-1R, U.S. Pat.No. 9,441,043), hPAM4 (anti-mucin, U.S. Pat. No. 7,282,567), hA20(anti-CD20, U.S. Pat. No. 7,151,164), hA19 (anti-CD19, U.S. Pat. No.7,109,304), hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLL1(anti-CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22, U.S. Pat. No.5,789,554), hMu-9 (anti-CSAp, U.S. Pat. No. 7,387,772), hL243(anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEACAM-5, U.S. Pat.No. 6,676,924), hMN-15 (anti-CEACAM-6, U.S. Pat. No. 8,287,865), hRS7(anti-EGP-1, U.S. Pat. No. 7,238,785), hMN-3 (anti-CEACAM-6, U.S. Pat.No. 7,541,440), Ab124 and Ab125 (anti-CXCR4, U.S. Pat. No. 7,138,496),the Examples section of each cited patent or application incorporatedherein by reference. More preferably, the antibody is IMMU-31(anti-AFP), hRS7 (anti-Trop-2), hMN-14 (anti-CEACAM-5), hMN-3(anti-CEACAM-6), hMN-15 (anti-CEACAM-6), hLL1 (anti-CD74), hLL2(anti-CD22), hL243 or IMMU-114 (anti-HLA-DR), hA19 (anti-CD19) or hA20(anti-CD20). As used herein, the terms epratuzumab and hLL2 areinterchangeable, as are the terms veltuzumab and hA20, hL243g4P,hL243gamma4P and IMMU-114. In a most preferred embodiment, the antibodyis an anti-Trop-2 antibody, such as hRS7, or an anti-HLA-DR antibody,such as hL243.

Alternative antibodies of use include, but are not limited to, abciximab(anti-glycoprotein IIb/IIIa), alemtuzumab (anti-CD52), bevacizumab(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab(anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab(anti-CD20), trastuzumab (anti-ErbB2), lambrolizumab (anti-PD-1receptor), atezolizumab (anti-PD-L1), MEDI4736 (anti-PD-L1), nivolumab(anti-PD-1 receptor), ipilimumab (anti-CTLA-4), abagovomab(anti-CA-125), adecatumumab (anti-EpCAM), atlizumab (anti-IL-6receptor), benralizumab (anti-CD125), obinutuzumab (GA101, anti-CD20),CC49 (anti-TAG-72), AB-PGI-XG1-026 (anti-PSMA, U.S. patent applicationSer. No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406), D2/B(anti-PSMA, WO 2009/130575), tocilizumab (anti-IL-6 receptor),basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab(anti-CD11a), GA101 (anti-CD20; Glycart Roche), muromonab-CD3 (anti-CD3receptor), natalizumab (anti-α4 integrin), omalizumab (anti-IgE);anti-TNF-α antibodies such as CDP571 (Ofei et al., 2011, Diabetes45:881-85), MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B, M303 (ThermoScientific, Rockford, Ill.), infliximab (Centocor, Malvern, Pa.),certolizumab pegol (UCB, Brussels, Belgium), anti-CD40L (UCB, Brussels,Belgium), adalimumab (Abbott, Abbott Park, Ill.), Benlysta (Human GenomeSciences); antibodies for therapy of Alzheimer's disease such as Alz 50(Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47), gantenerumab,solanezumab and infliximab; anti-fibrin antibodies like 59D8, T2G1s,MH1; anti-CD38 antibodies such as MOR03087 (MorphoSys AG), MOR202(Celgene), HuMax-CD38 (Genmab) or daratumumab (Johnson & Johnson);(anti-HIV antibodies such as P4/D10 (U.S. Pat. No. 8,333,971), Ab 75, Ab76, Ab 77 (Paulik et al., 1999, Biochem Pharmacol 58:1781-90), as wellas the anti-HIV antibodies described and sold by Polymun (Vienna,Austria), also described in U.S. Pat. Nos. 5,831,034, 5,911,989, andVcelar et al., AIDS 2007; 21(16):2161-2170 and Joos et al., Antimicrob.Agents Chemother. 2006; 50(5):1773-9, all incorporated herein byreference.

As discussed above, combination therapy with anti-HLA-DR antibody or ADCinvolves use of a kinase inhibitor, such as a Bruton's kinase inhibitor,e.g., ibrutinib (PCI-32765), PCI-45292, CC-292 (AVL-292), ONO-4059,GDC-0834, LFM-A13 or RN486, or a PI3K inhibitor, such as idelalisib,Wortmannin, demethoxyviridin, perifosine, PX-866, IPI-145 (duvelisib),BAY 80-6946, BEZ235, RP6530, TGR1202, SF1126, INK1117, GDC-0941, BKM120,XL147, XL765, Palomid 529, GSK1059615, ZSTK474, PWT33597, IC87114,TG100-115, CAL263, PI-103, GNE477, CUDC-907, AEZS-136 or LY294002.

However, in alternative embodiments, combination therapy may alsoinvolve administration of other drugs, either in unconjugated form orelse conjugated to a subject antibody. It is contemplated within thescope of the invention that an anti-HLA-DR antibody or immunoconjugate,in combination with a kinase inhibitor, may be administered along withone or more additional therapeutic agents. Where the additionaltherapeutic agent is an anti-cancer drug, it may be selected from thegroup consisting of 5-fluorouracil, afatinib, aplidin, azaribine,anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine,bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin,camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex,chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11),SN-38, carboplatin, cladribine, camptothecans, crizotinib,cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib,docetaxel, dactinomycin, daunorubicin, doxorubicin,2-pyrrolinodoxorubicine (2PDOX), pro-2PDOX, cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide, erlotinib,estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptorbinding agents, etoposide (VP16), etoposide glucuronide, etoposidephosphate, exemestane, fingolimod, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-O), fludarabine, flutamide, farnesyl-proteintransferase inhibitors, flavopiridol, fostamatinib, ganetespib,GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea, ibrutinib,idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase, lapatinib,lenolidamide, leucovorin, LFM-A13, lomustine, mechlorethamine,melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib,nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765,pentostatin, PSI-341, raloxifene, semustine, sorafenib, streptozocin,SU11248, sunitinib, tamoxifen, temazolomide (an aqueous form of DTIC),transplatinum, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine,vincristine, vinca alkaloids and ZD1839.

In other alternatives, anti-HLA-DR may be administered in combinationwith a PI3K inhibitor, such as idelalisib, Wortmannin, demethoxyviridin,perifosine, PX-866, IPI-145 (duvelisib), BAY 80-6946, BEZ235, RP6530,TGR1202, SF1126, INK1117, GDC-0941, BKM120, XL147, XL765, Palomid 529,GSK1059615, ZSTK474, PWT33597, IC87114, TG100-115, CAL263, PI-103,GNE477, CUDC-907, AEZS-136 or LY294002. Alternatively, the combinationtherapy may include use of a microtubule inhibitor, such as vincaalkaloids (e.g., vincristine, vinblastine), taxanes (e.g., paclitaxel),maytansinoids (e.g., mertansine) and auristatins. Other knownmicrotubule inhibitors include demecolcine, nocodazole, epothilone,docetaxel, discodermolide, colchicine, combrestatin, podophyllotoxin,CI-980, phenylahistins, steganacins, curacins, 2-methoxy estradiol,E7010, methoxy benzenesuflonamides, vinorelbine, vinflunine, vindesine,dolastatins, spongistatin, rhizoxin, tasidotin, halichondrins,hemiasterlins, cryptophycin 52, MMAE and eribulin mesylate (see, e.g.,Dumontet & Jordan, 2010, Nat Rev Drug Discov 9:790-803).

Where the anti-HLA-DR is an antibody-drug conjugate, such conjugateddrugs may be selected from camptothecin (CPT) and its analogs andderivatives and is more preferably SN-38. However, other drug moietiesthat may be utilized include taxanes (e.g., baccatin III, taxol),auristatins (e.g., MMAE), calicheamicins, epothilones, anthracyclines(e.g., doxorubicin (DOX), epirubicin, morpholinodoxorubicin(morpholino-DOX), cyanomorpholino-doxorubicin (cyanomorpholino-DOX),2-pyrrolinodoxorubicin (2-PDOX), a prodrug form of 2-PDOX (pro-2-PDOX),topotecan, etoposide, cisplatinum, oxaliplatin or carboplatin; see,e.g., Priebe W (ed.), ACS symposium series 574, published by AmericanChemical Society, Washington D.C., 1995 (332 pp) and Nagy et al., Proc.Natl. Acad. Sci. USA 93:2464-2469, 1996). Preferably, the antibody orfragment thereof links to at least one chemotherapeutic moiety;preferably 1 to about 5 drug moieties; more preferably 6 to about 12drug moieties, most preferably about 6 to 8 drug moieties. Where ananti-HLA-DR (hL243) antibody is conjugated to an SN-38 moiety, theconjugate may be referred to as IMMU-140.

An example of a water soluble CPT derivative is CPT-11. Extensiveclinical data are available concerning CPT-11's pharmacology and its invivo conversion to the active SN-38 (Iyer and Ratain, Cancer ChemotherPharmacol. 42:S31-43 (1998); Mathijssen et al., Clin Cancer Res.7:2182-2194 (2002); Rivory, Ann NY Acad Sci. 922:205-215, 2000)). Theactive form SN-38 is about 2 to 3 orders of magnitude more potent thanCPT-11. In specific preferred embodiments, the ADC may be anhMN-14-SN-38 (IMMU-130), hMN-3-SN-38, hMN-15-SN-38, IMMU-31-SN-38,hRS7-SN-38 (IMMU-132), hA20-SN-38, IMMU-140 (IMMU-140), hLL1-SN-38 orhLL2-SN-38 conjugate.

Various embodiments may concern use of the subject methods andcompositions to treat a cancer, including but not limited tonon-Hodgkin's lymphomas, B-cell acute and chronic lymphoid leukemias,Burkitt lymphoma, Hodgkin's lymphoma, acute large B-cell lymphoma, hairycell leukemia, acute myeloid leukemia, chronic myeloid leukemia, acutelymphocytic leukemia, chronic lymphocytic leukemia, T-cell lymphomas andleukemias, multiple myeloma, cancers of the skin, esophagus, stomach,colon, rectum, pancreas, lung, breast, ovary, bladder, endometrium,cervix, testes, kidney, liver, melanoma or other HLA-DR-producingtumors.

In certain embodiments involving treatment of cancer, the antibodies orADCs and kinase inhibitors may be used in combination with a standardanti-cancer therapy, such as surgery, radiation therapy, chemotherapy,immunotherapy with naked antibodies, including checkpoint-inhibitingantibodies, radioimmunotherapy, immunomodulators, vaccines, and thelike.

The claimed methods provide for shrinkage of solid tumors, inindividuals with previously resistant cancers, of 15% or more,preferably 20% or more, preferably 30% or more, more preferably 40% ormore in size (as measured by summing the longest diameter of targetlesions, as per RECIST or RECIST 1.1). The person of ordinary skill willrealize that tumor size may be measured by a variety of differenttechniques, such as total tumor volume, maximal tumor size in anydimension or a combination of size measurements in several dimensions.This may be with standard radiological procedures, such as computedtomography, magnet resonance imaging, ultrasonography, and/orpositron-emission tomography. The means of measuring size is lessimportant than observing a trend of decreasing tumor size withcombination therapy, preferably resulting in elimination of the tumor.However, to comply with RECIST guidelines, CT or MRI is preferred on aserial basis, and should be repeated to confirm measurements.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Comparative efficacies of anti-HLA-DR (IMMU-114) and anti-CD20(rituximab) unconjugated antibodies for treatment of CLL xenografts.Experiment was performed as disclosed in Example 1.

FIG. 2. Survival curves for CLL xenografted nude mice treated withunconjugated antibodies. Mice bearing JVM-3 xenografts were treated withunconjugated rituximab or IMMU-114, as disclosed in Example 1.

FIG. 3. Dose-response curves for IMMU-114 at constant ibrutinib.

FIG. 4. Dose-response curves for ibrutinib at constant IMMU-114.

FIG. 5. Isobologram showing synergistic effect of IMMU-114 incombination with a Bruton's tyrosine kinase inhibitor.

FIG. 6. Dose-response curves for idelalisib at constant IMMU-114.

FIG. 7. Isobologram for the PI3K inhibitor idelalisib in combinationwith IMMU-114.

FIG. 8. Survival curves comparing the efficacy of IMMU-114 vs.doxorubicin in nude mice with ALL xenografts.

FIG. 9. Survival curves for mice bearing disseminated acute myeloidleukemia xenografts, treated with IMMU-114 (hL243) vs. IMMU-140(hL243-SN-38).

FIG. 10. Survival curves for mice bearing disseminated acute lymphocyticleukemia xenografts, treated with IMMU-114 (hL243) vs. IMMU-140(hL243-SN-38).

FIG. 11. Survival curves for mice bearing disseminated multiple myelomaxenografts, treated with IMMU-114 (hL243) vs. IMMU-140 (hL243-SN-38).

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of theclaimed subject matter. Terms that are not expressly defined herein areused in accordance with their plain and ordinary meanings.

Unless otherwise specified, a or an means “one or more.”

The term about is used herein to mean plus or minus ten percent (10%) ofa value. For example, “about 100” refers to any number between 90 and110.

An antibody, as used herein, refers to a full-length (i.e., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an antigen-binding portion of an immunoglobulin molecule,such as an antibody fragment. An antibody or antibody fragment may beconjugated or otherwise derivatized within the scope of the claimedsubject matter. Such antibodies include but are not limited to IgG1,IgG2, IgG3, IgG4 (and IgG4 subforms), as well as IgA isotypes. As usedbelow, the abbreviation “MAb” may be used interchangeably to refer to anantibody, antibody fragment, monoclonal antibody or multispecificantibody.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv (single chain Fv), single domain antibodies(DABs or VHHs) and the like, including the half-molecules of IgG4 citedabove (van der Neut Kolfschoten et al. (Science 2007; 317(14September):1554-1557). Regardless of structure, an antibody fragment ofuse binds with the same antigen that is recognized by the intactantibody. The term “antibody fragment” also includes synthetic orgenetically engineered proteins that act like an antibody by binding toa specific antigen to form a complex. For example, antibody fragmentsinclude isolated fragments consisting of the variable regions, such asthe “Fv” fragments consisting of the variable regions of the heavy andlight chains and recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”). The fragments may be constructed in different ways toyield multivalent and/or multispecific binding forms.

A naked antibody is generally an entire antibody that is not conjugatedto a therapeutic agent. A naked antibody may exhibit therapeutic and/orcytotoxic effects, for example by Fc-dependent functions, such ascomplement fixation (CDC) and ADCC (antibody-dependent cellcytotoxicity). However, other mechanisms, such as apoptosis,anti-angiogenesis, anti-metastatic activity, anti-adhesion activity,inhibition of heterotypic or homotypic adhesion, and interference insignaling pathways, may also provide a therapeutic effect. Nakedantibodies include polyclonal and monoclonal antibodies, naturallyoccurring or recombinant antibodies, such as chimeric, humanized orhuman antibodies and fragments thereof. In some cases a “naked antibody”may also refer to a “naked” antibody fragment. As defined herein,“naked” is synonymous with “unconjugated,” and means not linked orconjugated to a therapeutic agent.

A chimeric antibody is a recombinant protein that contains the variabledomains of both the heavy and light antibody chains, including thecomplementarity determining regions (CDRs) of an antibody derived fromone species, preferably a rodent antibody, more preferably a murineantibody, while the constant domains of the antibody molecule arederived from those of a human antibody. For veterinary applications, theconstant domains of the chimeric antibody may be derived from that ofother species, such as a primate, cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a murine antibody, are transferred fromthe heavy and light variable chains of the murine antibody into humanheavy and light variable domains (framework regions). The constantdomains of the antibody molecule are derived from those of a humanantibody. In some cases, specific residues of the framework region ofthe humanized antibody, particularly those that are touching or close tothe CDR sequences, may be modified, for example replaced with thecorresponding residues from the original murine, rodent, subhumanprimate, or other antibody.

A human antibody is an antibody obtained, for example, from transgenicmice that have been “engineered” to produce human antibodies in responseto antigenic challenge. In this technique, elements of the human heavyand light chain loci are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for various antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, human antibody variable domain genes arecloned in-frame into either a major or minor coat protein gene of afilamentous bacteriophage, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. In this way, the phage mimics some of the properties of theB cell. Phage display can be performed in a variety of formats, fortheir review, see e.g. Johnson and Chiswell, Current Opinion inStructural Biology 3:5564-571 (1993). Human antibodies may also begenerated by in vitro activated B cells. See U.S. Pat. Nos. 5,567,610and 5,229,275, the Examples section of each of which is incorporatedherein by reference.

A therapeutic agent is an atom, molecule, or compound that is useful inthe treatment of a disease. Examples of therapeutic agents include, butare not limited to, antibodies, antibody fragments, ADCs, drugs,cytotoxic agents, pro-apopoptotic agents, toxins, nucleases (includingDNAses and RNAses), hormones, immunomodulators, chelators, boroncompounds, photoactive agents or dyes, radionuclides, oligonucleotides,interference RNA, siRNA, RNAi, anti-angiogenic agents, chemotherapeuticagents, cyokines, chemokines, prodrugs, enzymes, binding proteins orpeptides or combinations thereof.

An ADC is an antibody, antigen-binding antibody fragment, antibodycomplex or antibody fusion protein that is conjugated to at least onetherapeutic agent. Conjugation may be covalent or non-covalent.Preferably, conjugation is covalent.

As used herein, the term antibody fusion protein is arecombinantly-produced antigen-binding molecule in which one or morenatural antibodies, single-chain antibodies or antibody fragments arelinked to another moiety, such as a protein or peptide, a toxin, acytokine, a hormone, etc. In certain preferred embodiments, the fusionprotein may comprise two or more of the same or different antibodies,antibody fragments or single-chain antibodies fused together, which maybind to the same epitope, different epitopes on the same antigen, ordifferent antigens.

An immunomodulator is a therapeutic agent that when present, alters,suppresses or stimulates the body's immune system. Typically, animmunomodulator of use stimulates immune cells to proliferate or becomeactivated in an immune response cascade, such as macrophages, dendriticcells, B-cells, and/or T-cells. However, in some cases animmunomodulator may suppress proliferation or activation of immunecells. An example of an immunomodulator as described herein is acytokine, which is a soluble small protein of approximately 5-20 kDathat is released by one cell population (e.g., primed T-lymphocytes) oncontact with specific antigens, and which acts as an intercellularmediator between cells. As the skilled artisan will understand, examplesof cytokines include lymphokines, monokines, interleukins, and severalrelated signaling molecules, such as tumor necrosis factor (TNF) andinterferons. Chemokines are a subset of cytokines. Certain interleukinsand interferons are examples of cytokines that stimulate T cell or otherimmune cell proliferation. Exemplary interferons include interferon-α,interferon-β, interferon-γ and interferon-λ.

General Antibody Techniques

Techniques for preparing monoclonal antibodies against virtually anytarget antigen are well known in the art. See, for example, Köhler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991). Briefly, monoclonal antibodies can be obtained by injecting micewith a composition comprising an antigen, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A or Protein-G Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art, as discussedbelow.

The skilled artisan will realize that the claimed methods andcompositions may utilize any of a wide variety of antibodies known inthe art. Antibodies of use may be commercially obtained from a widevariety of known sources. For example, a variety of antibody secretinghybridoma lines are available from the American Type Culture Collection(ATCC, Manassas, Va.). A large number of antibodies against variousdisease targets, including but not limited to tumor-associated antigens,have been deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206′ 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953;5,525,338, the Examples section of each of which is incorporated hereinby reference. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art. Isolated antibodies may be conjugated totherapeutic agents that induce DNA strand breaks, such as camptothecinsor anthracyclines, using the techniques disclosed herein.

Chimeric and Humanized Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. Methods for constructingchimeric antibodies are well known in the art (e.g., Leung et al., 1994,Hybridoma 13:469).

A chimeric monoclonal antibody may be humanized by transferring themouse CDRs from the heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. The mouse framework regions (FR) in the chimeric monoclonalantibody are also replaced with human FR sequences. To preserve thestability and antigen specificity of the humanized monoclonal, one ormore human FR residues may be replaced by the mouse counterpartresidues. Humanized monoclonal antibodies may be used for therapeutictreatment of subjects. Techniques for production of humanized monoclonalantibodies are well known in the art. (See, e.g., Jones et al., 1986,Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen etal., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad.Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest etal., 1991, Biotechnology 9:266; Singer et al., J. Immun., 1993,150:2844.)

Other embodiments may concern non-human primate antibodies. Generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., WO 91/11465 (1991), and inLosman et al., Int. J. Cancer 46: 310 (1990). In another embodiment, anantibody may be a human monoclonal antibody. Such antibodies may beobtained from transgenic mice that have been engineered to producespecific human antibodies in response to antigenic challenge, asdiscussed below.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50; each incorporated herein by reference). Such fully humanantibodies are expected to exhibit even fewer side effects than chimericor humanized antibodies and to function in vivo as essentiallyendogenous human antibodies. In certain embodiments, the claimed methodsand procedures may utilize human antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as cancer (Dantas-Barbosa et al., 2005). Theadvantage to constructing human antibodies from a diseased individual isthat the circulating antibody repertoire may be biased towardsantibodies against disease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.) Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.) RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97,incorporated herein by reference). Library construction was performedaccording to Andris-Widhopf et al. (2000, In: Phage Display LaboratoryManual, Barbas et al. (eds), 1^(st) edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. pp. 9.1 to 9.22, incorporatedherein by reference). The final Fab fragments were digested withrestriction endonucleases and inserted into the bacteriophage genome tomake the phage display library. Such libraries may be screened bystandard phage display methods. The skilled artisan will realize thatthis technique is exemplary only and any known method for making andscreening human antibodies or antibody fragments by phage display may beutilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols as discussed above. Methods for obtaining humanantibodies from transgenic mice are described by Green et al., NatureGenet. 7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor etal., Int. Immun. 6:579 (1994). A non-limiting example of such a systemis the XENOMOUSE® (e.g., Green et al., 1999, J. Immunol. Methods231:11-23, incorporated herein by reference) from Abgenix (Fremont,Calif.). In the XENOMOUSE® and similar animals, the mouse antibody geneshave been inactivated and replaced by functional human antibody genes,while the remainder of the mouse immune system remains intact.

The XENOMOUSE® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH and Igkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XENOMOUSE®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XENOMOUSE®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XENOMOUSE® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Production of Antibody Fragments

Some embodiments of the claimed methods and/or compositions may concernantibody fragments. Such antibody fragments may be obtained, forexample, by pepsin or papain digestion of whole antibodies byconventional methods. For example, antibody fragments may be produced byenzymatic cleavage of antibodies with pepsin to provide a 5S fragmentdenoted F(ab′)₂. This fragment may be further cleaved using a thiolreducing agent and, optionally, a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce 3.5SFab′ monovalent fragments. Alternatively, an enzymatic cleavage usingpepsin produces two monovalent Fab fragments and an Fc fragment.Exemplary methods for producing antibody fragments are disclosed in U.S.Pat. Nos. 4,036,945; 4,331,647; Nisonoff et al., 1960, Arch. Biochem.Biophys., 89:230; Porter, 1959, Biochem. J., 73:119; Edelman et al.,1967, METHODS IN ENZYMOLOGY, page 422 (Academic Press), and Coligan etal. (eds.), 1991, CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley & Sons).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments or other enzymatic, chemical or genetic techniques also may beused, so long as the fragments bind to the antigen that is recognized bythe intact antibody. For example, Fv fragments comprise an associationof V_(H) and V_(L) chains. This association can be noncovalent, asdescribed in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA, 69:2659.Alternatively, the variable chains may be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde. SeeSandhu, 1992, Crit. Rev. Biotech., 12:437.

Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (scFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains, connected by an oligonucleotideslinker sequence. The structural gene is inserted into an expressionvector that is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are well-known in the art. See Whitlow et al., 1991, Methods: ACompanion to Methods in Enzymology 2:97; Bird et al., 1988, Science,242:423; U.S. Pat. No. 4,946,778; Pack et al., 1993, Bio/Technology,11:1271, and Sandhu, 1992, Crit. Rev. Biotech., 12:437.

Another form of an antibody fragment is a single-domain antibody (dAb),sometimes referred to as a single chain antibody. Techniques forproducing single-domain antibodies are well known in the art (see, e.g.,Cossins et al., Protein Expression and Purification, 2007, 51:253-59;Shuntao et al., Molec Immunol 2006, 43:1912-19; Tanha et al., J. Biol.Chem. 2001, 276:24774-780). Other types of antibody fragments maycomprise one or more complementarity-determining regions (CDRs). CDRpeptides (“minimal recognition units”) can be obtained by constructinggenes encoding the CDR of an antibody of interest. Such genes areprepared, for example, by using the polymerase chain reaction tosynthesize the variable region from RNA of antibody-producing cells. SeeLarrick et al., 1991, Methods: A Companion to Methods in Enzymology2:106; Ritter et al. (eds.), 1995, MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, pages 166-179 (CambridgeUniversity Press); Birch et al., (eds.), 1995, MONOCLONAL ANTIBODIES:PRINCIPLES AND APPLICATIONS, pages 137-185 (Wiley-Liss, Inc.)

Antibody Variations

In certain embodiments, the sequences of antibodies, such as the Fcportions of antibodies, may be varied to optimize the physiologicalcharacteristics of the conjugates, such as the half-life in serum.Methods of substituting amino acid sequences in proteins are widelyknown in the art, such as by site-directed mutagenesis (e.g. Sambrook etal., Molecular Cloning, A laboratory manual, 2^(nd) Ed, 1989). Inpreferred embodiments, the variation may involve the addition or removalof one or more glycosylation sites in the Fc sequence (e.g., U.S. Pat.No. 6,254,868, the Examples section of which is incorporated herein byreference). In other preferred embodiments, specific amino acidsubstitutions in the Fc sequence may be made (e.g., Hornick et al.,2000, J Nucl Med 41:355-62; Hinton et al., 2006, J Immunol 176:346-56;Petkova et al. 2006, Int Immunol 18:1759-69; U.S. Pat. No. 7,217,797;each incorporated herein by reference).

Target Antigens and Exemplary Antibodies

In a preferred embodiment, the antibody of use is targeted to the humanHLA-DR antigen. However, in alternative embodiments, antibodies may beused that recognize and/or bind to human antigens that are expressed athigh levels on target cells and that are expressed predominantly orexclusively on diseased cells versus normal tissues. More preferably,the antibodies internalize rapidly following binding. An exemplaryrapidly internalizing antibody is the LL1 (anti-CD74) antibody, with arate of internalization of approximately 8×10⁶ antibody molecules percell per day (e.g., Hansen et al., 1996, Biochem J. 320:293-300). Thus,a “rapidly internalizing” antibody may be one with an internalizationrate of about 1×10⁶ to about 1×10⁷ antibody molecules per cell per day.Antibodies of use in the claimed compositions and methods may includeMAbs with properties as recited above. Exemplary antibodies of use fortherapy of, for example, cancer include but are not limited to LL1(anti-CD74), LL2 or RFB4 (anti-CD22), veltuzumab (hA20, anti-CD20),rituxumab (anti-CD20), obinutuzumab (GA101, anti-CD20), lambrolizumab(anti-PD-1 receptor), nivolumab (anti-PD-1 receptor), ipilimumab(anti-CTLA-4), RS7 (anti-Trop-2), PAM4 (aka clivatuzumab, anti-MUC-5ac),MN-14 (anti-CEACAM-5), MN-15 or MN-3 (anti-CEACAM-6), Mu-9(anti-colon-specific antigen-p), Immu 31 (an anti-alpha-fetoprotein), R1(anti-IGF-1R), A19 (anti-CD19), TAG-72 (e.g., CC49), Tn, J591 or HuJ591(anti-PSMA (prostate-specific membrane antigen)), AB-PGI-XG1-026(anti-PSMA dimer), D2/B (anti-PSMA), G250 (an anti-carbonic anhydrase IXMAb), L243 (anti-HLA-DR) alemtuzumab (anti-CD52), bevacizumab(anti-VEGF), cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomabtiuxetan (anti-CD20); panitumumab (anti-EGFR); tositumomab (anti-CD20);and trastuzumab (anti-ErbB2). Such antibodies are known in the art(e.g., U.S. Pat. Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744;6,306,393; 6,653,104; 6,730.300; 6,899,864; 6,926,893; 6,962,702;7,074,403; 7,230,084; 7,238,785; 7,238,786; 7,256,004; 7,282,567;7,300,655; 7,312,318; 7,585,491; 7,612,180; 7,642,239; and U.S. PatentApplication Publ. No. 20050271671; 20060193865; 20060210475;20070087001; the Examples section of each incorporated herein byreference.) Specific known antibodies of use include hPAM4 (U.S. Pat.No. 7,282,567), hA20 (U.S. Pat. No. 7,151,164), hA19 (U.S. Pat. No.7,109,304), hIMMU-31 (U.S. Pat. No. 7,300,655), hLL1 (U.S. Pat. No.7,312,318,), hLL2 (U.S. Pat. No. 5,789,554), hMu-9 (U.S. Pat. No.7,387,772), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat. No.6,676,924), hMN-15 (U.S. Pat. No. 8,287,865), hR1 (U.S. patentapplication Ser. No. 14/061,1767), hRS7 (U.S. Pat. No. 7,238,785), hMN-3(U.S. Pat. No. 7,541,440), AB-PGI-XG1-026 (U.S. patent application Ser.No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO2009/130575) the text of each recited patent or application isincorporated herein by reference with respect to the Figures andExamples sections.

Other antigens that may be targeted include carbonic anhydrase IX, B7,CCL19, CCL21, CSAp, HER-2/neu, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5, CD8,CD11A, CD14, CD15, CD16, CD18, CD19, CD20 (e.g., C2B8, hA20, 1F5 MAbs),CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40,CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74,CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CEACAM-5,CEACAM-6, CTLA-4, alpha-fetoprotein (AFP), VEGF (e.g., AVASTIN®,fibronectin splice variant), ED-B fibronectin (e.g., L19), EGP-1(Trop-2), EGP-2 (e.g., 17-1A), EGF receptor (ErbB1) (e.g., ERBITUX®),ErbB2, ErbB3, Factor H, FHL-1, Flt-3, folate receptor, Ga 733, GRO-β,HMGB-1, hypoxia inducible factor (HIF), HM1.24, HER-2/neu, insulin-likegrowth factor (ILGF), IFN-γ, IFN-α, IFN-β, IFN-λ, IL-2R, IL-4R, IL-6R,IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17,IL-18, IL-25, IP-10, IGF-1R, Ia, HM1.24, gangliosides, HCG, the HLA-DRantigen to which L243 binds, CD66 antigens, i.e., CD66a-d or acombination thereof, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, macrophagemigration-inhibitory factor (MIF), MUC1, MUC2, MUC3, MUC4, MUC5ac,placental growth factor (P1GF), PSA (prostate-specific antigen), PSMA,PAM4 antigen, PD-1 receptor, NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1,Le(y), mesothelin, S100, tenascin, TAC, Tn antigen, Thomas-Friedenreichantigens, tumor necrosis antigens, tumor angiogenesis antigens, TNF-α,TRAIL receptor (RI and R2), Trop-2, VEGFR, RANTES, T101, as well ascancer stem cell antigens, complement factors C3, C3a, C3b, C5a, C5, andan oncogene product.

A comprehensive analysis of suitable antigen (Cluster Designation, orCD) targets on hematopoietic malignant cells, as shown by flow cytometryand which can be a guide to selecting suitable antibodies fordrug-conjugated immunotherapy, is Craig and Foon, Blood prepublishedonline Jan. 15, 2008; DOL 10.1182/blood-2007-11-120535.

The CD66 antigens consist of five different glycoproteins with similarstructures, CD66a-e, encoded by the carcinoembryonic antigen (CEA) genefamily members, BCG, CGM6, NCA, CGMI and CEA, respectively. These CD66antigens (e.g., CEACAM-6) are expressed mainly in granulocytes, normalepithelial cells of the digestive tract and tumor cells of varioustissues. Also included as suitable targets for cancers are cancer testisantigens, such as NY-ESO-1 (Theurillat et al., Int. J. Cancer 2007;120(11):2411-7), as well as CD79a in myeloid leukemia (Kozlov et al.,Cancer Genet. Cytogenet. 2005; 163(1):62-7) and also B-cell diseases,and CD79b for non-Hodgkin's lymphoma (Poison et al., Blood110(2):616-623). A number of the aforementioned antigens are disclosedin U.S. Provisional Application Ser. No. 60/426,379, entitled “Use ofMulti-specific, Non-covalent Complexes for Targeted Delivery ofTherapeutics,” filed Nov. 15, 2002. Cancer stem cells, which areascribed to be more therapy-resistant precursor malignant cellpopulations (Hill and Perris, J. Natl. Cancer Inst. 2007; 99:1435-40),have antigens that can be targeted in certain cancer types, such asCD133 in prostate cancer (Maitland et al., Ernst Schering Found. Sympos.Proc. 2006; 5:155-79), non-small-cell lung cancer (Donnenberg et al., J.Control Release 2007; 122(3):385-91), and glioblastoma (Beier et al.,Cancer Res. 2007; 67(9):4010-5), and CD44 in colorectal cancer (Dalerbaer al., Proc. Natl. Acad. Sci. USA 2007; 104(24)10158-63), pancreaticcancer (Li et al., Cancer Res. 2007; 67(3):1030-7), and in head and necksquamous cell carcinoma (Prince et al., Proc. Natl. Acad. Sci. USA 2007;104(3)973-8).

For multiple myeloma therapy, suitable targeting antibodies have beendescribed against, for example, CD38 and CD138 (Stevenson, Mol Med 2006;12(11-12):345-346; Tassone et al., Blood 2004; 104(12):3688-96), CD74(Stein et al., ibid.), CS1 (Tai et al., Blood 2008; 112(4):1329-37, andCD40 (Tai et al., 2005; Cancer Res. 65(13):5898-5906).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as cancers of thebladder, prostate, breast, lung, colon and chronic lymphocytic leukemia(e.g., Meyer-Siegler et al., 2004, BMC Cancer 12:34; Shachar & Haran,2011, Leuk Lymphoma 52:1446-54); autoimmune diseases such as rheumatoidarthritis and systemic lupus erythematosus (Morand & Leech, 2005, FrontBiosci 10:12-22; Shachar & Haran, 2011, Leuk Lymphoma 52:1446-54);kidney diseases such as renal allograft rejection (Lan, 2008, NephronExp Nephrol. 109:e79-83); and numerous inflammatory diseases(Meyer-Siegler et al., 2009, Mediators Inflamm epub Mar. 22, 2009;Takahashi et al., 2009, Respir Res 10:33; Milatuzumab (hLL1) is anexemplary anti-CD74 antibody of therapeutic use for treatment ofMIF-mediated diseases.

Anti-TNF-α antibodies are known in the art and may be of use to treatimmune diseases, such as autoimmune disease, immune dysfunction (e.g.,graft-versus-host disease, organ transplant rejection) or diabetes.Known antibodies against TNF-α include the human antibody CDP571 (Ofeiet al., 2011, Diabetes 45:881-85); murine antibodies MTNFAI, M2TNFAI,M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, Ill.);infliximab (Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,Belgium); and adalimumab (Abbott, Abbott Park, Ill.). These and manyother known anti-TNF-α antibodies may be used in the claimed methods andcompositions. Other antibodies of use for therapy of immunedysregulatory or autoimmune disease include, but are not limited to,anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab orhL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25);daclizumab (anti-CD25); efalizumab (anti-CD11a); muromonab-CD3 (anti-CD3receptor); anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-α4integrin) and omalizumab (anti-IgE).

In another preferred embodiment, antibodies are used that internalizerapidly and are then re-expressed, processed and presented on cellsurfaces, enabling continual uptake and accretion of circulatingconjugate by the cell. An example of a most-preferred antibody/antigenpair is LL1, an anti-CD74 MAb (invariant chain, class II-specificchaperone, Ii) (see, e.g., U.S. Pat. Nos. 6,653,104; 7,312,318; theExamples section of each incorporated herein by reference). The CD74antigen is highly expressed on B-cell lymphomas (including multiplemyeloma) and leukemias, certain T-cell lymphomas, melanomas, colonic,lung, and renal cancers, glioblastomas, and certain other cancers (Onget al., Immunology 98:296-302 (1999)). A review of the use of CD74antibodies in cancer is contained in Stein et al., Clin Cancer Res. 2007Sep. 15; 13(18 Pt 2):55565-5563s, incorporated herein by reference.

The diseases that are preferably treated with anti-CD74 antibodiesinclude, but are not limited to, non-Hodgkin's lymphoma, Hodgkin'sdisease, melanoma, lung, renal, colonic cancers, glioblastomemultiforme, histiocytomas, myeloid leukemias, and multiple myeloma.Continual expression of the CD74 antigen for short periods of time onthe surface of target cells, followed by internalization of the antigen,and re-expression of the antigen, enables the targeting LL1 antibody tobe internalized along with any drug moiety it carries. This allows ahigh, and therapeutic, concentration of LL1-drug conjugate to beaccumulated inside such cells. Internalized LL1-drug conjugates arecycled through lysosomes and endosomes, and the drug moiety is releasedin an active form within the target cells.

Antibodies of use to treat autoimmune disease or immune systemdysfunctions (e.g., graft-versus-host disease, organ transplantrejection) are known in the art and may be conjugated to SN-38 using thedisclosed methods and compositions. Antibodies of use to treatautoimmune/immune dysfunction disease may bind to exemplary antigensincluding, but not limited to, BCL-1, BCL-2, BCL-6, CD1a, CD2, CD3, CD4,CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16, CD19, CD20,CD21, CD22, CD23, CD25, CD33, CD34, CD38, CD40, CD40L, CD41a, CD43,CD45, CD55, CD74, TNF-alpha, interferon and HLA-DR. Antibodies that bindto these and other target antigens, discussed above, may be used totreat autoimmune or immune dysfunction diseases. Autoimmune diseasesthat may be treated with antibodies or ADCs may include acute idiopathicthrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura,dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupuserythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, ANCA-associated vasculitides, Addison's disease, rheumatoidarthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

The antibodies discussed above and other known antibodies againstdisease-associated antigens may be used as CPT-conjugates, morepreferably SN-38-conjugates, in the practice of the claimed methods andcompositions.

Bispecific and Multispecific Antibodies

Bispecific antibodies are useful in a number of biomedical applications.For instance, a bispecific antibody with binding sites for a tumor cellsurface antigen and for a T-cell surface receptor can direct the lysisof specific tumor cells by T cells. Bispecific antibodies recognizinggliomas and the CD3 epitope on T cells have been successfully used intreating brain tumors in human patients (Nitta, et al. Lancet. 1990;355:368-371). A preferred bispecific antibody is an anti-CD3 X anti-CD19antibody. In alternative embodiments, an anti-CD3 antibody or fragmentthereof may be attached to an antibody or fragment against anotherB-cell associated antigen, such as anti-CD3 X anti-CD20, anti-CD3 Xanti-CD22, anti-CD3 X anti-HLA-DR or anti-CD3 X anti-CD74. In certainembodiments, the subject anti-HLA-DR antibody may be utilized incombination with a bispecific antibody as disclosed herein.

Numerous methods to produce bispecific or multispecific antibodies areknown, as disclosed, for example, in U.S. Pat. No. 7,405,320, theExamples section of which is incorporated herein by reference.Bispecific antibodies can be produced by the quadroma method, whichinvolves the fusion of two different hybridomas, each producing amonoclonal antibody recognizing a different antigenic site (Milstein andCuello, Nature, 1983; 305:537-540).

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies (Staerz, et al. Nature, 1985; 314:628-631; Perez,et al. Nature, 1985; 316:354-356). Bispecific antibodies can also beproduced by reduction of each of two parental monoclonal antibodies tothe respective half molecules, which are then mixed and allowed toreoxidize to obtain the hybrid structure (Staerz and Bevan. Proc NatlAcad Sci USA. 1986; 83:1453-1457). Another alternative involveschemically cross-linking two or three separately purified Fab′ fragmentsusing appropriate linkers. (See, e.g., European Patent Application0453082).

Other methods include improving the efficiency of generating hybridhybridomas by gene transfer of distinct selectable markers viaretrovirus-derived shuttle vectors into respective parental hybridomas,which are fused subsequently (DeMonte, et al. Proc Natl Acad Sci USA.1990, 87:2941-2945); or transfection of a hybridoma cell line withexpression plasmids containing the heavy and light chain genes of adifferent antibody.

Cognate V_(H) and V_(L) domains can be joined with a peptide linker ofappropriate composition and length (usually consisting of more than 12amino acid residues) to form a single-chain Fv (scFv) with bindingactivity. Methods of manufacturing scFvs are disclosed in U.S. Pat. Nos.4,946,778 and 5,132,405, the Examples section of each of which isincorporated herein by reference. Reduction of the peptide linker lengthto less than 12 amino acid residues prevents pairing of V_(H) and V_(L)domains on the same chain and forces pairing of V_(H) and V_(L) domainswith complementary domains on other chains, resulting in the formationof functional multimers. Polypeptide chains of V_(H) and V_(L) domainsthat are joined with linkers between 3 and 12 amino acid residues formpredominantly dimers (termed diabodies). With linkers between 0 and 2amino acid residues, trimers (termed triabody) and tetramers (termedtetrabody) are favored, but the exact patterns of oligomerization appearto depend on the composition as well as the orientation of V-domains(V_(H)-linker-V_(L) or V_(L)-linker-V_(H)), in addition to the linkerlength.

These techniques for producing multispecific or bispecific antibodiesexhibit various difficulties in terms of low yield, necessity forpurification, low stability or the labor-intensiveness of the technique.More recently, a technique known as “dock and lock” (DNL) has beenutilized to produce combinations of virtually any desired antibodies,antibody fragments and other effector molecules (see, e.g., U.S. Pat.Nos. 7,521,056; 7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,858,070;7,871,622; 7,906,121; 7,906,118; 8,163,291; 7,901,680; 7,981,398;8,003,111 and 8,034,352, the Examples section of each of whichincorporated herein by reference). The technique utilizes complementaryprotein binding domains, referred to as anchoring domains (AD) anddimerization and docking domains (DDD), which bind to each other andallow the assembly of complex structures, ranging from dimers, trimers,tetramers, quintamers and hexamers. These form stable complexes in highyield without requirement for extensive purification. The DNL techniqueallows the assembly of monospecific, bispecific or multispecificantibodies. Any of the techniques known in the art for making bispecificor multispecific antibodies may be utilized in the practice of thepresently claimed methods.

DOCK-AND-LOCK® (DNL®)

In preferred embodiments, a bispecific or multispecific antibody isformed as a DOCK-AND-LOCK® (DNL®) complex (see, e.g., U.S. Pat. Nos.7,521,056; 7,527,787; 7,534,866; 7,550,143; 7,666,400; 7,858,070;7,871,622; 7,906,121; 7,906,118; 8,163,291; 7,901,680; 7,981,398;8,003,111 and 8,034,352, the Examples section of each of which isincorporated herein by reference.) Generally, the technique takesadvantage of the specific and high-affinity binding interactions thatoccur between a dimerization and docking domain (DDD) sequence of theregulatory (R) subunits of cAMP-dependent protein kinase (PKA) and ananchor domain (AD) sequence derived from any of a variety of AKAPproteins (Baillie et al., FEBS Letters. 2005; 579: 3264. Wong and Scott,Nat. Rev. Mol. Cell Biol. 2004; 5: 959). The DDD and AD peptides may beattached to any protein, peptide or other molecule. Because the DDDsequences spontaneously dimerize and bind to the AD sequence, thetechnique allows the formation of complexes between any selectedmolecules that may be attached to DDD or AD sequences.

Although the standard DNL® complex comprises a trimer with twoDDD-linked molecules attached to one AD-linked molecule, variations incomplex structure allow the formation of dimers, trimers, tetramers,pentamers, hexamers and other multimers. In some embodiments, the DNL®complex may comprise two or more antibodies, antibody fragments orfusion proteins which bind to the same antigenic determinant or to twoor more different antigens. The DNL® complex may also comprise one ormore other effectors, such as proteins, peptides, immunomodulators,cytokines, interleukins, interferons, binding proteins, peptide ligands,carrier proteins, toxins, ribonucleases such as onconase, inhibitoryoligonucleotides such as siRNA, antigens or xenoantigens, polymers suchas PEG, enzymes, therapeutic agents, hormones, cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents or any other molecule oraggregate.

PKA, which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (RI and RII), and eachtype has α and β isoforms (Scott, Pharmacol. Ther. 1991; 50:123). Thus,the four isoforms of PKA regulatory subunits are RIα, RIβ, RIIα andRIIβ. The R subunits have been isolated only as stable dimers and thedimerization domain has been shown to consist of the first 44amino-terminal residues of RIIα (Newlon et al., Nat. Struct. Biol. 1999;6:222). As discussed below, similar portions of the amino acid sequencesof other regulatory subunits are involved in dimerization and docking,each located near the N-terminal end of the regulatory subunit. Bindingof cAMP to the R subunits leads to the release of active catalyticsubunits for a broad spectrum of serine/threonine kinase activities,which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for Rildimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIcc, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human PKAregulatory subunits and the AD of AKAP as an excellent pair of linkermodules for docking any two entities, referred to hereafter as A and B,into a noncovalent complex, which could be further locked into a DNL®complex through the introduction of cysteine residues into both the DDDand AD at strategic positions to facilitate the formation of disulfidebonds. The general methodology of the approach is as follows. Entity Ais constructed by linking a DDD sequence to a precursor of A, resultingin a first component hereafter referred to as a. Because the DDDsequence would effect the spontaneous formation of a dimer, A would thusbe composed of a₂. Entity B is constructed by linking an AD sequence toa precursor of B, resulting in a second component hereafter referred toas b. The dimeric motif of DDD contained in a₂ will create a dockingsite for binding to the AD sequence contained in b, thus facilitating aready association of a₂ and b to form a binary, trimeric complexcomposed of a₂b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsshould bring the reactive thiol groups placed onto both the DDD and ADinto proximity (Chmura et al., Proc. Natl. Acad. Sci. USA. 2001;98:8480) to ligate site-specifically. Using various combinations oflinkers, adaptor modules and precursors, a wide variety of DNL®constructs of different stoichiometry may be produced and used (see,e.g., U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNL®construct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

In various embodiments, an antibody or antibody fragment may beincorporated into a DNL® complex by, for example, attaching a DDD or ADmoiety to the C-terminal end of the antibody heavy chain, as describedin detail below. In more preferred embodiments, the DDD or AD moiety,more preferably the AD moiety, may be attached to the C-terminal end ofthe antibody light chain (see, e.g., U.S. patent application Ser. No.13/901,737, filed May 24, 2013, the Examples section of which isincorporated herein by reference.)

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Id.). It has beenreported that G1m1 antibodies contain allotypic sequences that tend toinduce an immune response when administered to non-G1m1 (nG1m1)recipients, such as G1m3 patients (Id.). Non-G1m1 allotype antibodiesare not as immunogenic when administered to G1m1 patients (Id.).

The human G1m1 allotype comprises the amino acids aspartic acid at Kabatposition 356 and leucine at Kabat position 358 in the CH3 sequence ofthe heavy chain IgG1. The nG1m1 allotype comprises the amino acidsglutamic acid at Kabat position 356 and methionine at Kabat position358. Both G1m1 and nG1m1 allotypes comprise a glutamic acid residue atKabat position 357 and the allotypes are sometimes referred to as DELand EEM allotypes. A non-limiting example of the heavy chain constantregion sequences for G1m1 and nG1m1 allotype antibodies is shown for theexemplary antibodies rituximab (SEQ ID NO:7) and veltuzumab (SEQ IDNO:8).

Rituximab heavy chain variable region sequence (SEQ ID NO: 7)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Veltuzumab heavy chain variable region(SEQ ID NO: 8) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variationscharacteristic of IgG allotypes and their effect on immunogenicity. Theyreported that the G1m3 allotype is characterized by an arginine residueat Kabat position 214, compared to a lysine residue at Kabat 214 in theG1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acidat Kabat position 356, methionine at Kabat position 358 and alanine atKabat position 431. The G1m1,2 allotype was characterized by asparticacid at Kabat position 356, leucine at Kabat position 358 and glycine atKabat position 431. In addition to heavy chain constant region sequencevariants, Jefferis and Lefranc (2009) reported allotypic variants in thekappa light chain constant region, with the Km1 allotype characterizedby valine at Kabat position 153 and leucine at Kabat position 191, theKm1,2 allotype by alanine at Kabat position 153 and leucine at Kabatposition 191, and the Km3 allotypoe characterized by alanine at Kabatposition 153 and valine at Kabat position 191.

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies. Table 1compares the allotype sequences of rituximab vs. veltuzumab. As shown inTable 1, rituximab (G1m17,1) is a DEL allotype IgG1, with an additionalsequence variation at Kabat position 214 (heavy chain CH1) of lysine inrituximab vs. arginine in veltuzumab. It has been reported thatveltuzumab is less immunogenic in subjects than rituximab (see, e.g.,Morchhauser et al., 2009, J Clin Oncol 27:3346-53; Goldenberg et al.,2009, Blood 113:1062-70; Robak & Robak, 2011, BioDrugs 25:13-25), aneffect that has been attributed to the difference between humanized andchimeric antibodies. However, the difference in allotypes between theEEM and DEL allotypes likely also accounts for the lower immunogenicityof veltuzumab.

TABLE 1 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes Complete 214 356/358 431 allotype (allotype)(allotype) (allotype) Rituximab G1m17, 1 K 17 D/L 1 A — Veltuzumab G1m3R 3 E/M — A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the G1m3 allotype, characterized byarginine at Kabat 214, and the nG1m1,2 null-allotype, characterized byglutamic acid at Kabat position 356, methionine at Kabat position 358and alanine at Kabat position 431. Surprisingly, it was found thatrepeated subcutaneous administration of G1m3 antibodies over a longperiod of time did not result in a significant immune response. Inalternative embodiments, the human IgG4 heavy chain in common with theG1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356,methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicityappears to relate at least in part to the residues at those locations,use of the human IgG4 heavy chain constant region sequence fortherapeutic antibodies is also a preferred embodiment. Combinations ofG1m3 IgG1 antibodies with IgG4 antibodies may also be of use fortherapeutic administration.

Amino Acid Substitutions

In alternative embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. For example, the DDD and/or ADsequences used to make DNL® constructs may be modified as discussedabove.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Conjugation Protocols

The preferred conjugation protocol is based on a thiol-maleimide, athiol-vinylsulfone, a thiol-bromoacetamide, or a thiol-iodoacetamidereaction that is facile at neutral or acidic pH. This obviates the needfor higher pH conditions for conjugations as, for instance, would benecessitated when using active esters. Further details of exemplaryconjugation protocols are described below in the Examples section.

Therapeutic Treatment

In another aspect, the invention relates to a method of treating asubject, preferably a human subject, comprising administering atherapeutically effective amount of an antibody or ADC as describedherein to a subject, preferably in combination with a Bruton's kinaseinhibitor and/or PI3K inhibitor. Diseases that may be treated with theantibodies or ADCs described herein include, but are not limited toB-cell malignancies (e.g., non-Hodgkin's lymphoma, mantle cell lymphoma,multiple myeloma, Hodgkin's lymphoma, diffuse large B cell lymphoma,Burkitt lymphoma, follicular lymphoma, acute lymphocytic leukemia,chronic lymphocytic leukemia, hairy cell leukemia) using, for example ananti-HLA-DR antibody such as the IMMU-114. Other diseases include, butare not limited to, cancers of the skin, esophagus, stomach, colon,rectum, pancreas, lung, breast, ovary, bladder, endometrium, cervix,testes, kidney, liver, melanoma or other HLA-DR-producing tumors. Inalternative embodiments involving treatment with an antibody binding toa TAA other than HLA-DR, the person of ordinary skill will realize thatdifferent types of cancer that are known to express the TAA may betreated.

Such therapeutics can be given once or repeatedly, depending on thedisease state and tolerability of the conjugate, and can also be usedoptionally in combination with other therapeutic modalities, such assurgery, external radiation, radioimmunotherapy, immunotherapy,chemotherapy, antisense therapy, interference RNA therapy, gene therapy,and the like. Each combination will be adapted to the tumor type, stage,patient condition and prior therapy, and other factors considered by themanaging physician.

As used herein, the term “subject” refers to any animal (i.e.,vertebrates and invertebrates) including, but not limited to mammals,including humans. It is not intended that the term be limited to aparticular age or sex. Thus, adult and newborn subjects, as well asfetuses, whether male or female, are encompassed by the term. Dosesgiven herein are for humans, but can be adjusted to the size of othermammals, as well as children, in accordance with weight or square metersize. Where an antibody or ADC is administered to a human subject, theperson of ordinary skill will realize that the target antigen to whichthe antibody or ADC binds will be a human antigen.

In preferred embodiments, antibodies or ADCs comprising an anti-HLA-DRMAb, such as hL243 (IMMU-114), can be used to treat lymphoma, leukemia,multiple myeloma, cancers of the skin, esophagus, stomach, colon,rectum, pancreas, lung, breast, ovary, bladder, endometrium, cervix,testes, kidney, liver, melanoma or other HLA-DR-producing tumors, asdisclosed in U.S. Pat. No. 7,612,180, the Examples section of which isincorporated herein by reference. An hL243 antibody is a humanizedantibody comprising the heavy chain CDR sequences CDR1 (NYGMN, SEQ IDNO:1), CDR2 (WINTYTREPTYADDFKG, SEQ ID NO:2), and CDR3 (DITAVVPTGFDY,SEQ ID NO:3) and light chain CDR sequences CDR1 (RASENIYSNLA, SEQ IDNO:4), CDR2 (AASNLAD, SEQ ID NO:5), and CDR3 (QHFWTTPWA, SEQ ID NO:6).

In another preferred embodiment, the antibodies or ADCs can be used totreat autoimmune disease or immune system dysfunction (e.g.,graft-versus-host disease, organ transplant rejection). Antibodies ofuse to treat autoimmune/immune dysfunction disease may bind to exemplaryantigens including, but not limited to, BCL-1, BCL-2, BCL-6, CD1a, CD2,CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD15, CD16,CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD34, CD38, CD40, CD40L,CD41a, CD43, CD45, CD55, CD56, CCD57, CD59, CD64, CD71, CD74, CD79a,CD79b, CD117, CD138, FMC-7 and HLA-DR. Antibodies that bind to these andother target antigens, discussed above, may be used to treat autoimmuneor immune dysfunction diseases. Autoimmune diseases that may be treatedwith antibodies or ADCs may include acute idiopathic thrombocytopenicpurpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, diabetes mellitus, Henoch-Schonlein purpura,post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,ANCA-associated vasculitides, Addison's disease, rheumatoid arthritis,multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

In another preferred embodiment, a therapeutic agent used in combinationwith the subject antibodies may comprise one or more isotopes.Radioactive isotopes useful for treating diseased tissue include, butare not limited to—¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁷Cu, ⁹⁰Y,¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy,¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr,⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²²⁷Th and²¹¹Pb. The therapeutic radionuclide preferably has a decay-energy in therange of 20 to 6,000 keV, preferably in the ranges 60 to 200 keV for anAuger emitter, 100-2,500 keV for a beta emitter, and 4,000-6,000 keV foran alpha emitter. Maximum decay energies of usefulbeta-particle-emitting nuclides are preferably 20-5,000 keV, morepreferably 100-4,000 keV, and most preferably 500-2,500 keV. Alsopreferred are radionuclides that substantially decay with Auger-emittingparticles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109,In-111, Sb-119, I-125, Ho-161, Os-189m and Ir-192. Decay energies ofuseful beta-particle-emitting nuclides are preferably <1,000 keV, morepreferably <100 keV, and most preferably <70 keV. Also preferred areradionuclides that substantially decay with generation ofalpha-particles. Such radionuclides include, but are not limited to:Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221,At-217, Bi-213, Th-227 and Fm-255. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-10,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.Additional potential radioisotopes of use include ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br,¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru,¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm,¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co,⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

Radionuclides and other metals may be delivered, for example, usingchelating groups attached to an antibody or ADC. Macrocyclic chelatessuch as NOTA, DOTA, and TETA are of use with a variety of metals andradiometals, most particularly with radionuclides of gallium, yttriumand copper, respectively. Such metal-chelate complexes can be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelates, such as macrocyclic polyethers for complexing ²²³Ra,may be used.

Therapeutic agents of use in combination with the antibodies or ADCsdescribed herein also include, for example, chemotherapeutic drugs suchas vinca alkaloids, anthracyclines, epidophyllotoxins, taxanes,antimetabolites, tyrosine kinase inhibitors, alkylating agents,antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic andproapoptotic agents, particularly doxorubicin, methotrexate, taxol,other camptothecins, and others from these and other classes ofanticancer agents, and the like. Other cancer chemotherapeutic drugsinclude nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes,folic acid antagonists, pyrimidine analogs, purine analogs, platinumcoordination complexes, hormones, and the like. Suitablechemotherapeutic agents are described in REMINGTON'S PHARMACEUTICALSCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN ANDGILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillanPublishing Co. 1985), as well as revised editions of these publications.Other suitable chemotherapeutic agents, such as experimental drugs, areknown to those of skill in the art.

Exemplary drugs of use include, but are not limited to, 5-fluorouracil,afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib,AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib,bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin(CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin,cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine,dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin,daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2PDOX), pro-2PDOX,cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicinglucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib,entinostat, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, exemestane, fingolimod,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, farnesyl-protein transferase inhibitors, flavopiridol,fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine,hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib,L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine,vinblastine, vincristine, vinca alkaloids and ZD1839. Such agents may bepart of the conjugates described herein or may alternatively beadministered in combination with the disclosed antibodies or ADCs,either prior to, simultaneously with or after the antibody or ADC.Alternatively, one or more therapeutic naked antibodies as are known inthe art may be used in combination with the disclosed antibodies orADCs. Exemplary therapeutic naked antibodies are described above.

In preferred embodiments, a therapeutic agent to be used in combinationan antibody or ADC and/or a Bruton's kinase or PI3K inhibitor may be amicrotubule inhibitor, such as a vinca alkaloid, a taxanes, amaytansinoid or an auristatin. Exemplary known microtubule inhibitorsinclude paclitaxel, vincristine, vinblastine, mertansine, epothilone,docetaxel, discodermolide, combrestatin, podophyllotoxin, CI-980,phenylahistins, steganacins, curacins, 2-methoxy estradiol, E7010,methoxy benzenesuflonamides, vinorelbine, vinflunine, vindesine,dolastatins, spongistatin, rhizoxin, tasidotin, halichondrins,hemiasterlins, cryptophycin 52, MMAE and eribulin mesylate.

In an alternative embodiment, a therapeutic agent to be used incombination with an antibody or ADC and/or a Bruton's kinase or PI3Kinhibitor, is a PARP inhibitor, such as olaparib, talazoparib (BMN-673),rucaparib, veliparib, CEP 9722, MK 4827, BGB-290, ABT-888, AG014699,BSI-201, CEP-8983 or 3-aminobenzamide.

As discussed above, Bruton's kinase inhibitors include, but are notlimited to, ibrutinib (PCI-32765), PCI-45292, CC-292 (AVL-292),ONO-4059, GDC-0834, LFM-A13 or RN486.

As discussed above, PI3K inhibitors include, but are not limited to,idelalisib, Wortmannin, demethoxyviridin, perifosine, PX-866, IPI-145(duvelisib), BAY 80-6946, BEZ235, RP6530, TGR1202, SF1126, INK1117,GDC-0941, BKM120, XL147, XL765, Palomid 529, GSK1059615, ZSTK474,PWT33597, IC87114, TG100-115, CAL263, PI-103, GNE477, CUDC-907, AEZS-136or LY294002.

Therapeutic agents that may be used in concert with the antibodies orADCs also may comprise toxins conjugated to targeting moieties. Toxinsthat may be used in this regard include ricin, abrin, ribonuclease(RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviralprotein, gelonin, diphtheria toxin, Pseudomonas exotoxin, andPseudomonas endotoxin. (See, e.g., Pastan. et al., Cell (1986), 47:641,and Sharkey and Goldenberg, CA Cancer J Clin. 2006 July-August;56(4):226-43.) Additional toxins suitable for use herein are known tothose of skill in the art and are disclosed in U.S. Pat. No. 6,077,499.

Yet another class of therapeutic agent may comprise one or moreimmunomodulators. Immunomodulators of use may be selected from acytokine, a stem cell growth factor, a lymphotoxin, an hematopoieticfactor, a colony stimulating factor (CSF), an interferon (IFN),erythropoietin, thrombopoietin and a combination thereof. Specificallyuseful are lymphotoxins such as tumor necrosis factor (TNF),hematopoietic factors, such as interleukin (IL), colony stimulatingfactor, such as granulocyte-colony stimulating factor (G-CSF) orgranulocyte macrophage-colony stimulating factor (GM-CSF), interferon,such as interferons-α, -β, -γ or -λ, and stem cell growth factor, suchas that designated “S1 factor”. Included among the cytokines are growthhormones such as human growth hormone, N-methionyl human growth hormone,and bovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -ß; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-ß; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-ß; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand lymphotoxin (LT). As used herein, the term cytokine includesproteins from natural sources or from recombinant cell culture andbiologically active equivalents of the native sequence cytokines.

Chemokines of use include RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10.

The person of ordinary skill will realize that the subject antibodies orADCs may be used alone or in combination with one or more othertherapeutic agents, such as a second antibody, second antibody fragment,second ADC, radionuclide, toxin, drug, chemotherapeutic agent, radiationtherapy, chemokine, cytokine, immunomodulator, enzyme, hormone,oligonucleotide, RNAi or siRNA. Preferably, the therapeutic agent is aBruton's kinase inhibitor or a PI3K inhibitor.

Formulation and Administration

Suitable routes of administration of the antibodies, ADCs and/or kinaseinhibitors include, without limitation, parenteral, subcutaneous,rectal, transmucosal, intestinal administration, intramuscular,intramedullary, intrathecal, direct intraventricular, intravenous,intravitreal, intraperitoneal, intranasal, or intraocular injections.The preferred routes of administration are parenteral. Alternatively,one may administer the compound in a local rather than systemic manner,for example, via injection of the compound directly into a solid tumoror by subcutaneous administration. Certain therapeutic agents, such asmicrotubule inhibitors, PARP inhibitors, Bruton's kinase inhibitors orPI3K inhibitors may be designed to be administered orally.

Antibodies or ADCs can be formulated according to known methods toprepare pharmaceutically useful compositions, whereby the antibody orADC is combined in a mixture with a pharmaceutically suitable excipient.Sterile phosphate-buffered saline is one example of a pharmaceuticallysuitable excipient. Other suitable excipients are well-known to those inthe art. See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS ANDDRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro(ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (MackPublishing Company 1990), and revised editions thereof.

In a preferred embodiment, the antibody or ADC is formulated in Good'sbiological buffer (pH 6-7), using a buffer selected from the groupconsisting of N-(2-acetamido)-2-aminoethanesulfonic acid (ACES);N-(2-acetamido)iminodiacetic acid (ADA);N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES);4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES);2-(N-morpholino)ethanesulfonic acid (MES);3-(N-morpholino)propanesulfonic acid (MOPS);3-(N-morpholinyl)-2-hydroxypropanesulfonic acid (MOPSO); andpiperazine-N,N′-bis(2-ethanesulfonic acid) [Pipes]. More preferredbuffers are MES or MOPS, preferably in the concentration range of 20 to100 mM, more preferably about 25 mM. Most preferred is 25 mM MES, pH6.5. The formulation may further comprise 25 mM trehalose and 0.01% v/vpolysorbate 80 as excipients, with the final buffer concentrationmodified to 22.25 mM as a result of added excipients. The preferredmethod of storage is as a lyophilized formulation of the conjugates,stored in the temperature range of −20° C. to 2° C., with the mostpreferred storage at 2° C. to 8° C.

The antibody or ADC can be formulated for intravenous administrationvia, for example, bolus injection, slow infusion or continuous infusion.Preferably, the antibody of the present invention is infused over aperiod of less than about 4 hours, and more preferably, over a period ofless than about 3 hours. For example, the first 25-50 mg could beinfused within 30 minutes, preferably even 15 min, and the remainderinfused over the next 2-3 hrs. Formulations for injection can bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

In another preferred embodiment, an antibody or ADC may be administeredby subcutaneous injection, either as a primary treatment or formaintenance therapy following, e.g., intravenous administration of theantibody or ADC.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic conjugate. Control releasepreparations can be prepared through the use of polymers to complex oradsorb the ADC. For example, biocompatible polymers include matrices ofpoly(ethylene-co-vinyl acetate) and matrices of a polyanhydridecopolymer of a stearic acid dimer and sebacic acid. Sherwood et al.,Bio/Technology 10: 1446 (1992). The rate of release of an antibody orADC from such a matrix depends upon the molecular weight, the amount ofantibody or ADC within the matrix, and the size of dispersed particles.Saltzman et al., Biophys. J. 55: 163 (1989); Sherwood et al., supra.Other solid dosage forms are described in Ansel et al., PHARMACEUTICALDOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18thEdition (Mack Publishing Company 1990), and revised editions thereof.

Generally, the dosage of an administered antibody or ADC for humans willvary depending upon such factors as the patient's age, weight, height,sex, general medical condition and previous medical history. It may bedesirable to provide the recipient with a dosage of ADC that is in therange of from about 1 mg/kg to 24 mg/kg, more preferably 4 to 16 mg/kg,more preferably 8 to 10 mg/kg, as a single intravenous infusion,although a lower or higher dosage also may be administered ascircumstances dictate. A dosage of 1-20 mg/kg for a 70 kg patient, forexample, is 70-1,400 mg, or 41-824 mg/m² for a 1.7-m patient. The dosagemay be repeated as needed, for example, once per week for 4-10 weeks,once per week for 8 weeks, or once per week for 4 weeks. It may also begiven less frequently, such as every other week for several months, ormonthly or quarterly for many months, as needed in a maintenancetherapy. Preferred dosages may include, but are not limited to, 1 mg/kg,2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg,10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 22 mg/kg and 24 mg/kg. However, inalternative embodiments, wherein the ADC is administered subcutaneously,preferred dosages would be in the range of 1.5 to 4.0 mg/kg. Asubcutaneous dosage may be administered at a single site, or mayalternatively be administered at multiple sites, each of which mayreceive a dosage of 1.5 to 4.0 mg/kg. In certain preferred embodiments,an initial treatment of ADC administered intravenously may be followedby a maintenance dosage administered subcutaneously.

The dosage is preferably administered multiple times, once or twice aweek, or as infrequently as once every 3 or 4 weeks. A minimum dosageschedule of 4 weeks, more preferably 8 weeks, more preferably 16 weeksor longer may be used. The schedule of administration may compriseadministration once or twice a week, on a cycle selected from the groupconsisting of: (i) weekly; (ii) every other week; (iii) one week oftherapy followed by two, three or four weeks off; (iv) two weeks oftherapy followed by one, two, three or four weeks off; (v) three weeksof therapy followed by one, two, three, four or five week off; (vi) fourweeks of therapy followed by one, two, three, four or five week off;(vii) five weeks of therapy followed by one, two, three, four or fiveweek off; (viii) monthly and (ix) every 3 weeks. The cycle may berepeated 2, 4, 6, 8, 10, 12, 16 or 20 times or more.

Alternatively, an antibody or ADC may be administered as one dosageevery 2 or 3 weeks, repeated for a total of at least 3 dosages. Or,twice per week for 4-6 weeks. If the dosage is lowered to approximately200-300 mg/m² (340 mg per dosage for a 1.7-m patient, or 4.9 mg/kg for a70 kg patient), it may be administered once or even twice weekly for 4to 10 weeks. Alternatively, the dosage schedule may be decreased, namelyevery 2 or 3 weeks for 2-3 months. It has been determined, however, thateven higher doses, such as 12 mg/kg once weekly or once every 2-3 weekscan be administered by slow i.v. infusion, for repeated dosing cycles.The dosing schedule can optionally be repeated at other intervals anddosage may be given through various parenteral routes, with appropriateadjustment of the dose and schedule.

In preferred embodiments, the antibodies or ADCs are of use for therapyof cancer. Examples of cancers include, but are not limited to,carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia,myeloma, or lymphoid malignancies. More particular examples of suchcancers are noted below and include: squamous cell cancer (e.g.,epithelial squamous cell cancer), Ewing sarcoma, Wilms tumor,astrocytomas, glioblastomas, lung cancer including small-cell lungcancer, non-small cell lung cancer, adenocarcinoma of the lung andsquamous carcinoma of the lung, cancer of the peritoneum, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer,bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrinetumors, medullary thyroid cancer, differentiated thyroid carcinoma,breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrialcancer or uterine carcinoma, salivary gland carcinoma, kidney or renalcancer, prostate cancer, vulvar cancer, anal carcinoma, penilecarcinoma, as well as head-and-neck cancer. The term “cancer” includesprimary malignant cells or tumors (e.g., those whose cells have notmigrated to sites in the subject's body other than the site of theoriginal malignancy or tumor) and secondary malignant cells or tumors(e.g., those arising from metastasis, the migration of malignant cellsor tumor cells to secondary sites that are different from the site ofthe original tumor).

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia,Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult SoftTissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, AnalCancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer,Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the RenalPelvis and Ureter, Central Nervous System (Primary) Lymphoma, CentralNervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma,Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood(Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia,Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, ChildhoodCerebellar Astrocytoma, Childhood Cerebral Astrocytoma, ChildhoodExtracranial Germ Cell Tumors, Childhood Hodgkin's Disease, ChildhoodHodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma,Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, ChildhoodNon-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Primary Liver Cancer, ChildhoodRhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood VisualPathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, EndocrinePancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma,Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and RelatedTumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor,Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer,Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, GastricCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, GermCell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Headand Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma,Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers,Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell PancreaticCancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders,Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, MalignantThymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic OccultPrimary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer,Metastatic Squamous Neck Cancer, Multiple Myeloma, MultipleMyeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, MyelogenousLeukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavityand Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma,Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell LungCancer, Occult Primary Metastatic Squamous Neck Cancer, OropharyngealCancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant FibrousHistiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone,Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian LowMalignant Potential Tumor, Pancreatic Cancer, Paraproteinemias,Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary LiverCancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvisand Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary GlandCancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small CellLung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenström's macroglobulinemia, Wilms' tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used totreat malignant or premalignant conditions and to prevent progression toa neoplastic or malignant state, including but not limited to thosedisorders described above. Such uses are indicated in conditions knownor suspected of preceding progression to neoplasia or cancer, inparticular, where non-neoplastic cell growth consisting of hyperplasia,metaplasia, or most particularly, dysplasia has occurred (for review ofsuch abnormal growth conditions, see Robbins and Angell, BasicPathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be treated include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be treated include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps oradenomas, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

In preferred embodiments, the method of the invention is used to inhibitgrowth, progression, and/or metastasis of cancers, in particular thoselisted above.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias; e.g., acute lymphocytic leukemia, acute myelocytic leukemia[including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia]) and chronic leukemias (e.g., chronic myelocytic[granulocytic] leukemia and chronic lymphocytic leukemia), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, and retinoblastoma.

Autoimmune diseases that may be treated with antibodies or ADCs mayinclude acute and chronic immune thrombocytopenias, dermatomyositis,Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus,lupus nephritis, rheumatic fever, polyglandular syndromes, bullouspemphigoid, diabetes mellitus, Henoch-Schonlein purpura,post-streptococcal nephritis, erythema nodosum, Takayasu's arteritis,ANCA-associated vasculitides, Addison's disease, rheumatoid arthritis,multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitis obliterans,Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, bullous pemphigoid,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis, psoriasis or fibrosing alveolitis.

Kits

Various embodiments may concern kits containing components suitable fortreating diseased tissue in a patient. Exemplary kits may contain atleast one antibody or ADC as described herein. A kit may also include akinase inhibitor selected from Bruton's kinase inhibitors and/or PI3Kinhibitors. If the composition containing components for administrationis not formulated for delivery via the alimentary canal, such as by oraldelivery, a device capable of delivering the kit components through someother route may be included. One type of device, for applications suchas parenteral delivery, is a syringe that is used to inject thecomposition into the body of a subject. Inhalation devices may also beused.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES

Various embodiments of the present invention are illustrated by thefollowing examples, without limiting the scope thereof.

Example 1 Combination Therapy with Anti-HLA-DR Antibody and Bruton'sKinase or Phosphoinositide-3-kinase (PI3K) Inhibitors in CLL and ALL

IMMU-114 is a humanized anti-HLA-DR IgG₄ monoclonal antibody currentlyunder investigation for non-Hodgkin's lymphoma and CLL(ClinicalTrials.gov, NCT01728207). The present Example compared IMMU-114efficacy to anti-CD20 or doxorubicin therapy, respectively, as well incombination with Bruton's tyrosine kinase (Btk) orphosphoinositide-3-kinase (PI3K) inhibitors. The combination therapy isof use for treating hematopoietic cancers, such as ALL or CLL.

Materials and Methods

The human CLL cell line, JVM-3, was grown s.c. in SCID mice. Once tumorsreached ˜0.2 cm³, they were divided into treatment groups of eitherIMMU-114 or rituximab (200, 100, or 50 mg, twice weekly for 4 weeks).Study survival endpoint was tumor progression to >1.0 cm³. In vitro,JMV-3 was treated with various concentrations of either a Btk inhibitor(ibrutinib) or PI3K inhibitor (idelalisib) in the presence of a constantamount of IMMU-114. IC₅₀-values were determined, data were normalized,and isobolograms generated for each inhibitor to determine overalleffect. For ALL, MN-60 cells were injected i.v. into SCID mice. After 5days, animals received IMMU-114 (50 or 25 mg, 2×weekly for 4 weeks) ordoxorubicin (3×20 mg qdx3d induction phase, followed by a 60-mg bolusinjection maintenance phase on week 3). Disease progression was declaredupon the onset of hind-limb paralysis.

Results

As discussed below, mice with JVM-3 (CLL) tumors had a median survivaltime (MST) of 14 days for saline controls, while therapy with rituximabsignificantly improved survival (P<0.0102); the MST was only 19 days forthe two highest doses. In contrast, mice treated with IMMU-114 had aMST >42 days for all three doses tested (P<0.0001), providing an overallsuperior tumor growth control over rituximab (P<0.0116). In vitro, anadditive effect was observed in JVM-3 when IMMU-114 was combined witheither ibrutinib or idelalisib. In the ALL disseminated MN-60 diseasemodel, mice were refractory to the doxorubicin treatment, succumbing todisease at the same rate as saline controls (MST=23 and 21 days,respectively). Importantly, IMMU-114, at both the 50 and 25 mg doses,provided a significant survival benefit compared to both saline controland doxorubicin-treated animals (MST>39 days, P<0.0001). IMMU-114therapy was well tolerated in all these studies, as evidenced by nosignificant loss in weight.

Significant anti-tumor effects of IMMU-114, compared to rituximab, wereobserved in mice bearing CLL xenograft tumors (JVM-3). JVM-3 human CLLcells were harvested from tissue culture and analyzed by FACS for HLA-DR(alpha chain) and CD20 surface expression (not shown). Mean fluorescenceintensity (MFI) histograms of HLA-DR, CD20, and secondary backgrounddemonstrated similar expression levels of both HLA-DR and CD20 (notshown). FIG. 1 shows the relative efficacies of IMMU-114 (anti-HLA-DR)vs. rituximab (anti-CD20) in CLL xenografts. C.B.-17 SCID mice wereinjected s.c. with JVM-3 cells. Once tumor volumes (TV) reached ˜0.2cm³, animals were divided into treatment groups as indicated on thegraph. IMMU-114 had significant anti-tumor effects compared to saline orrituximab at every dose tested (P<0.0116; AUC) (FIG. 1).

The anti-tumor effect of anti-HLA-DR mAb translated into a greater than2-fold improvement in survival (end-point of TV>1.0 cm³) compared torituximab treated mice (P<0.0107) or 3-fold compared to saline control(P<0.0001), even at doses as low as 50 mg (HED=0.23 mg/kg) (FIG. 2).Mice bearing JVM-3 CLL xenografts exhibited median survival of 14 days(saline control) vs. 18-19 days when treated with rituxumab (50 to 200μg) or 42-54 days when treated with IMMU-114 (50 to 200 μg).

The survival data for CLL-bearing nude mice treated with IMMU-114 orrituximab at 14 to 18 days of treatment is summarized in Table 2,showing an area under the curve survival comparison for the two groups.At each dosage, IMMU-114 was significantly more efficacious thanrituximab. At the highest dosage of 200 mg/ml, the P-value (AUC) was0.0004. Thus, there was a highly significant difference in survival inmice treated with IMMU-114 compared to rituximab.

TABLE 2 Area under the curve comparison between IMMU-114 and rituximabfor CLL. Area under the curve comparisons between IMMU-114 and Rituximabtreated JVM-3 tumor-bearing mice. Time of Tumor Volumes (cm³) P-ValueTreatments Comparison On that day (mean ± s.d.) (AUC) IMMU-114 RituximabUp to therapy 0.347 ± 0.120 vs. 0.0004 (200 μg) (200 μg) Day 18 1.017 ±0.266 IMMU-114 Rituximab Up to therapy 0.242 ± 0.101 vs. 0.0116 (100 μg)(100 μg) Day 14 0.643 ± 0.286 IMMU-114 Rituximab Up to therapy 0.270 ±0.094 vs. 0.0039 (50 μg) (50 μg) Day 14 0.765 ± 0.307

The significantly improved efficacy of IMMU-114 vs. rituxumab continuedto the end of the treatment, as shown in Table 3. At termination of thestudy, mice treated with IMMU-114 still showed significantly improvedsurvival at all dosages when compared to rituximab-treated mice. At thehighest dosage of 200 μg (equivalent to 10 mg/kg for a 20 g mouse), themedian survival was 46 days for IMMU-114 treated mice, compared to 18days for rituximab-treated mice (P-value<0.0001).

TABLE 3 Survival cares comparing efficacy of IMMU-114 (anti-HLA-DR) vs.rituximab (anti-CD20) Survival curve comparisons between IMMU-114 andRituximab treated JVM-3 tumor-bearing mice. Median Survival P-ValueTreatments Times (log-rank) IMMU-114 Rituximab (200 μg) 46 days versus18 days <0.0001 (200 μg) IMMU-114 Rituximab (100 μg) 54 days versus 18days 0.0107 (100 μg) IMMU-114 Rituximab (50 μg) 42 days versus 19 days<0.0001 (50 μg)

Synergistic Effect of Anti-HLA-DR Antibody and Bruton's KinaseInhibitors

Combination therapy with the IMMU-114 antibody and the exemplaryBruton's tyrosine kinase inhibitor ibrutinib was observed in CLL (JVM-3)xenografts. Dose/response curves for each agent alone were first testedto determine single agent IC₁₀, IC₂₀, or IC₃₀-values after a 96-hincubation (FIG. 3, FIG. 4). In combination assays, IMMU-114 was testedin JVM-3 across a range of concentrations (i.e., dose/response curves)(FIG. 3). One set of wells only received IMMU-114, while another setreceived IMMU-114 as dose/response with a constant amount of ibrutinib(e.g., IC₁₀-concentration) (FIG. 3). Two other sets used ibrutinib atIC₂₀ or IC₃₀ concentrations (FIG. 3). For each IMMU-114 dose/responsecurve, the IC₅₀-value was determined from these data. As shown in FIG.3, the IC₅₀ values for IMMU-114 ranged from 1.89×10⁻⁹ M in the absenceof ibrutinib to 1.66×10⁻⁹ M in 0.25 μM ibrutinib, 0.66×10⁻⁹ M in 0.5 μMibrutinib, and 0.20×10⁻⁹ M in 1 μM ibrutinib.

A similar set of experiments was performed, varying the dosage ofibrutinib tested in the presence of constant amounts of IMMU-114 (i.e.,IC₁₀-, IC20-, or IC₃₀-concentrations) (FIG. 4). As shown in FIG. 4, theIC₅₀ value for ibrutinib varied from 2.37^(×10−6M) in the absence ofIMMU-114, to 0.94×10⁻⁶M in 0.25 nM IMMU-114, 0.48×10⁻⁶M in 0.50 nMIMMU-114 and 0.37×10⁻⁶ M in 0.65 nM IMMU-114. (Note that hL243designates the same antibody as IMMU-114.)

These changes in IC₅₀-values were normalized and plotted as isobolograms(FIG. 5). Each combination demonstrates a synergistic interactionbetween IMMU-114 and the Bruton's kinase inhibitor, ibrutinib.

Additive Effect of Anti-HLA-DR Antibody and Bruton's Kinase Inhibitors

The Figures discusssed below show the additive growth inhibitory effectsof IMMU-114 in combination with an exemplary PI3K inhibitor (idelalisib)in CLL (JVM-3) cells. Dose/response curves for IMMU-114 were firsttested to determine IC₁₀-, IC₂₀-, or IC₃₀-values after a 96-hincubation. In combination assays, idelalisib was tested in the JVM-3cell line across a range of concentrations (i.e., dose/response curves)(FIG. 6). One set of wells only received idelalisib, while another setreceived idelalisib as dose/response with a constant amount of IMMU-114(e.g., ˜IC₁₀-concentration) (FIG. 6). Two other sets use IMMU-114 atIC₂₀ or IC₃₀ concentrations (FIG. 6). For each idelalisib dose/responsecurve, the IC₅₀ value was determined from these graphed data, at eachconcentration of IMMU-114. The values of IC₅₀ for idelalisib determinedranged from 14.26 μM in the absence of IMMU-114 to 8.28 μM in 0.25 nMIMMU-114, 6.0 μM in 0.5 nM IMMU-114 to 4.45 μM in 0.65 nM IMMU-114.

An isobologram of normalized IC₅₀-values from two different experimentsdemonstrated an additive effect for the combination of IMMU-114 withidelalisib in this human CLL cell line (FIG. 7).

Superior Efficacy of IMMU-114 Compared to Doxorubicin Treatment in ALLCells

The superior efficacy of anti-HLA-DR antibody compared to standardanti-cancer treatments was shown vs. doxorubicin in refractory human ALLcells (MN-60). Human ALL cells (MN-60) were harvested from tissueculture and injected i.v. into C.B.-17 SCID mice. Animals wererandomized into groups of 10 mice each. Five days later therapy began asindicated on FIG. 8. Doxorubicin therapy was designed to model clinicaluse of this drug, i.e., an induction phase consisting of the MTD of 60mg was fractionated over three consecutive days (20 mg each day)followed by a maintenance dose of 60 mg three weeks later.

Mice treated with doxorubicin alone did not respond to treatment andwere no different from saline control (FIG. 8). Conversely, both dosesof IMMU-114 of 25 or 50 μg provided ˜2-fold improvement in survivalcompared to all control groups. Additionally, there was a dose/responseobserved in that mice treated with 50 mg IMMU-114 had a significantsurvival benefit in comparison to mice treated with 25 mg (P=0.0336).

Conclusions

IMMU-114 is a humanized anti-HLA-DR IgG₄ antibody that has shownencouraging clinical responses in patients with non-Hodgkin's lymphomaand CLL (ClinicalTrials.gov, NCT01728207). As shown above, in a CLLpreclinical disease model that has similar HLA-DR and CD20 expression,IMMU-114 provided a significantly better anti-tumor effect and improvedsurvival benefit compared to rituximab, even at doses as low as 50 mg(HED=0.23 mg/kg).

In vitro, the combination of IMMU-114 and two different kinaseinhibitors was evaluated in CLL. IMMU-114 and the Bruton's kinaseinhibitor, ibrutinib, combined to provide a synergistic growthinhibitory interaction, whereas the combination with a PI3K inhibitor,idelalisib, resulted in an additive effect.

In a doxorubicin-refractory ALL model, IMMU-114 therapy provided asignificant 2-fold improvement in survival at doses as low as 25 mg(HED=0.12 mg/kg).

These data show the efficacy of IMMU-114 therapy for human patients inthese and other diverse hematopoietic cancers alone and combined withcurrent chemotherapeutic agents that are active in their respectiveindications. The surprising synergistic effect of anti-HLA-DR mAbs incombination with other anti-cancer agents provides the basis forimproved efficacy and decreased toxicity for treating humanhematopoietic cancers.

The person of ordinary skill will realize that these effects are notlimited to the specific anti-HLA-DR antibody, Bruton's tyrosine kinaseinhibitor or phosphoinositide-3-kinase (PI3K) inhibitor used, but mayalso be applied with other anti-HLA-DR antibodies or and/kinaseinhibitors.

Example 2 Treatment of Relapsed Chronic Lymphocytic Leukemia withIMMU-114 and Ibrutinib

A 65-year-old woman with a history of CLL, as defined by theInternational Workshop on Chronic Lymphocytic Leukemia and World HealthOrganization classifications, presents with relapsed disease after priortherapies with fludarabine, dexamethasone, and rituximab, as well as aregimen of CVP. She now has fever and night sweats associated withgeneralized lymph node enlargement, a reduced hemoglobin and plateletproduction, as well as a rapidly rising leukocyte count. Her LDH iselevated and the beta-2-microglobulin is almost twice normal. Thepatient is given therapy with anti-HLA-DR IMMU-114 (IgG4 hL243) at 200mg administered i.v. twice weekly. The antibody was given in combinationwith the Bruton's kinase inhibitor ibrutinib, administered orally at adosage of 420 mg daily. After 6 weeks, evaluation of the patient'shematological and lab values indicate that she has shown a partialresponse to the combination therapy.

Example 3 Treatment of Relapsed/Refractory Non-Hodgkin's Lymphoma (NHL)with IMMU-114 and Idelalisib

Seventeen patients with follicular NHL who have not shown a response torituximab and an alkylating agent, or relapsed within 6 months afterreceipt of those therapies receive 200 mg IMMU-114, injected s.c. twiceweekly, in combination with the PI3K inhibitor idelalisib, administeredorally at 150 mg twice daily, until the disease progresses or thepatient withdraws from the study. Responses are assessed by CT scans,with other evaluations including adverse events, B-cell blood levels,serum IMMU-114 levels and human anti-IMMU-114 (HAHA) titers.

Only occasional, mild to moderate transient injection reactions are seenand no other safety issues except neutropenia up to Grade 3 (butreversible after interrupting therapy until reduced to Grade 1) areobserved. Transient B-cell depletion (up to about 25%) is observed. Theobjective response rate (partial responses plus complete responses pluscomplete responses unconfirmed) is 47% (8/17) with a completeresponse/complete response unconfirmed rate of 24% (4/17). Four of theeight objective responses continue for 30 weeks or more. All serumsamples evaluated for human anti-IMMU-114 antibody (HAHA) are negative.

Example 4 Preparation of CL2A-SN-38

To the mixture of commercially available Fmoc-Lys(MMT)-OH (0.943 g),p-aminobenzyl alcohol (0.190 g) in methylene choloride (10 mL) was addedEEDQ (0.382 g) at room temperature and stirred for 4 h. Extractive workup followed by flash chromatograph yielded 1.051 g of material as whitefoam. All HPLC analyses were performed by Method B as stated in‘General’ in section 0148. HPLC ret. time: 3.53 min., Electrospray massspectrum showed peaks at m/e 745.8 (M+H) and m/e 780.3 (M+Cl⁻),consistent with structure. This intermediate (0.93 g) was dissolved indiethylamine (10 mL) and stirred for 2 h. After solvent removal, theresidue was washed in hexane to obtain 0.6 g of the intermediate ((2) inScheme-1) as colorless precipitate (91.6% pure by HPLC). HPLC ret. time:2.06 min. Electrospray mass spectrum showed peaks at m/e 523.8 (M+H),m/e 546.2 (M+Na) and m/e 522.5 (M−H).

This crude intermediate (0.565 g) was coupled with commerciallyavailableO-(2-azidoethyl)-O′—(N-diglycolyl-2-aminoethyl)heptaethyleneglycol('PEG-N3′, 0.627 g) using EEDQ in methylene chloride (10 mL). Solventremoval and flash chromatography yielded 0.99 g of the product ((3) inScheme-1; light yellow oil; 87% yield). HPLC ret. time: 2.45 min.Electrospray mass spectrum showed peaks at m/e 1061.3 (M+H), m/e 1082.7(M+Na) and m/e 1058.8 (M−H), consistent with structure. Thisintermediate (0.92 g) was reacted with10-O-TBDMS-SN-38-20-O-chloroformate ((5) in Scheme-1) in methylenechloride (10 mL) for 10 min under argon. The mixture was purified byflash chromatography to obtain 0.944 g as light yellow oil ((6) inScheme-1; yield=68%). HPLC ret. time: 4.18 min. To this intermediate(0.94 g) in methylene chloride (10 mL) was added the mixture of TBAF (1Min THF, 0.885 mL) and acetic acid (0.085 mL) in methylene chloride (3mL), then stirred for 10 min. The mixture was diluted with methylenechloride (100 mL), washed with 0.25 M sodium citrate and brine. Thesolvent removal yielded 0.835 g of yellow oily product. HPLC ret. time:2.80 min., (99% purity). Electrospray mass spectrum showed peaks at m/e1478 (M+H), m/e 1500.6 (M+Na), m/e 1476.5 (M−H), m/e 1590.5 (M+TFA),consistent with structure.

This azido-derivatized SN-38 intermediate (0.803 g) was reacted with4-(N-maleimidomethyl)-N-(2-propynyl)cyclohexane-1-carboxamide (0.233 g)in methylene chloride (10 mL) in presence of CuBr (0.0083 g,), DIEA(0.01 mL) and triphenylphosphine (0.015 g), for 18 h. Extractive workup, including washing with and 0.1M EDTA (10 mL), and flashchromatography yielded 0.891 g as yellow foam. (yield=93%), HPLC ret.time: 2.60 min. Electrospray mass spectrum showed peaks at m/e 1753.3(M+H), m/e 1751.6 (M−H), 1864.5 (M+TFA), consistent with structure.Finally, deprotection of the penultimate intermediate (0.22 g) with amixture of dichloroacetic acid (0.3 mL) and anisole (0.03 mL) inmethylene chloride (3 mL), followed by precipitation with ether yielded0.18 g (97% yield) of CL2A-SN-38; (7) in Scheme-3) as light yellowpowder. HPLC ret. time: 1.88 min. Electrospray mass spectrum showedpeaks at m/e 1480.7 (M+H), 1478.5 (M−H), consistent with structure.

Example 5 Conjugation of Bifunctional SN-38 Products to Mildly ReducedAntibodies

The anti-CEACAM-5 humanized MAb, hMN-14 (also known as labetuzumab), theanti-CD22 humanized MAb, hLL2 (also known as epratuzumab), the anti-CD20humanized MAb, hA20 (also known as veltuzumab), the anti-EGP-1 humanizedMAb, hRS7, and anti-mucin humanized MAb, hPAM4 (also known asclivatuzumab), were used in these studies. Each antibody was reducedwith dithiothreitol (DTT), used in a 50-to-70-fold molar excess, in 40mM PBS, pH 7.4, containing 5.4 mM EDTA, at 37° C. (bath) for 45 min. Thereduced product was purified by size-exclusion chromatography and/ordiafiltration, and was buffer-exchanged into a suitable buffer at pH6.5. The thiol content was determined by Ellman's assay, and was in the6.5-to-8.5 SH/IgG range. Alternatively, the antibodies were reduced withTris (2-carboxyethyl) phosphine (TCEP) in phosphate buffer at pH in therange of 5-7, followed by in situ conjugation. The reduced MAb wasreacted with ˜10-to-15-fold molar excess of CL2A-SN-38 using DMSO at7-15% v/v as co-solvent, and incubating for 20 min at ambienttemperature. The conjugate was purified by centrifuged SEC, passagethrough a hydrophobic column, and finally byultrafiltration-diafiltration. The product was assayed for SN-38 byabsorbance at 366 nm and correlating with standard values, while theprotein concentration was deduced from absorbance at 280 nm, correctedfor spillover of SN-38 absorbance at this wavelength. This way, theSN-38/MAb substitution ratios were determined. The purified conjugateswere stored as lyophilized formulations in glass vials, capped undervacuum and stored in a −20° C. freezer. SN-38 molar substitution ratios(MSR) obtained for some of these conjugates, which were typically in the5-to-7 range, are shown in Table 4. The person of ordinary skill willrealize that the conjugation method may be applied to any antibody ofuse in the disclosed methods.

TABLE 4 SN-38/MAb Molar substitution ratios (MSR) in some conjugates MAbConjugate MSR hMN-14 hMN-14-CL2A-SN-38 6.1 hRS7 hRS7-CL2A-SN-38 5.8 hA20hA20-CL2A-SN-38 5.8 hLL2 hLL2-CL2A-SN-38 5.7 hPAM4 hPAM4-CL2A-SN-38 5.9

Example 6 Treatment of Relapsed Chronic Lymphocytic Leukemia withIMMU-140 (IMMU-140) and Idelalisib

A 67-year-old man with a history of CLL, as defined by the InternationalWorkshop on Chronic Lymphocytic Leukemia and World Health Organizationclassifications, presents with relapsed disease after prior therapieswith fludarabine, dexamethasone, and rituximab, as well as a regimen ofCVP. He now has fever and night sweats associated with generalized lymphnode enlargement, a reduced hemoglobin and platelet production, as wellas a rapidly rising leukocyte count. His LDH is elevated and thebeta-2-microglobulin is almost twice normal. The patient is giventherapy with anti-HLA-DR IMMU-140 (IgG4 IMMU-140) conjugate incombination with idelalisib on a 21-day cycle. The IMMU-140 isadministered i.v. at 6 mg/kg on days 1 and 8 and idelalisib isadministered at a dosage of 100 mg p.o. on days 1, 7 and 14 of thecycle, then the cycle is repeated. After 4 cycles, evaluation shows thatthe patient's hematological parameters improve and his circulating CLLcells appear to be decreasing in number. The therapy is resumed foranother 3 cycles, after which his hematological and lab values indicatethat he has a partial response.

Example 7 Treatment of Follicular Lymphoma Patient with IMMU-140,Rucaparib and Paclitaxel

A 60-year-old male presents with abdominal pain and the presence of apalpable mass. The patient has CT and FDG-PET studies confirming thepresence of the mass with pathologic adenopathies in the mediastinum,axillary, and neck nodes. Lab tests are unremarkable except for elevatedLDH and beta-2-microglobulin. Bone marrow biopsy discloses severalparatrabecular and perivascular lymphoid aggregates. The final diagnosisis grade-2 follicular lymphoma, stage IVA, with a FLIPI score of 4. Thelongest diameter of the largest involved node is 7 cm. The patient isgiven combination therapy with a humanized anti-HLA-DR-SN-38 conjugate(IMMU-140), plus rucaparib and paclitaxel, on a 21-day cycle. The ADC isgiven at 6 mg/kg on days 7 and 14, rucaparib is administered at 10 mg/m²on days 1, 8 and 15, and paclitaxel is administered at 125 mg/m² on days1, 7 and 14 of the cycle. After 5 cycles, bone marrow and imaging (CT)evaluations show a partial response, where the measurable lesionsdecrease by about 60% and the bone marrow is much less infiltrated.Also, LDH and beta-2-microglobulin titers also decrease.

Example 8 Treatment of Relapsed Precursor B-cell ALL with IMMU-140 PlusOlaparib

This 51-year-old woman has been under therapy for precursor,Philadelphia chromosome-negative, B-cell ALL. The patient has receivedprior therapy with clofarabine and cytarabine, resulting in considerablehematological toxicity, but no response. A course of high-dosecytarabine (ara-C) was also started, but could not be tolerated by thepatient. She is given IMMU-140 and olaparib on a 21-day cycle, withdoses of IMMU-140 by i.v. infusion of 6 mg/kg on days 7 and 14 andolaparib at 200 mg twice a day p.o. on days 1-10 of the cycle.

After 3 cycles, surprisingly, she shows improvement in her blood andmarrow counts, sufficient for a partial response to be determined. Aftera rest of 2 months because of neutropenia (Grade 3), therapy resumes foranother 4 courses. At this time, she is much improved and is underconsideration for maintenance therapy to try to bring her to a stagewhere she could be a candidate for stem-cell transplantation.

Example 9 Treatment of Non-Hodgkin's Lymphoma with IMMU-114 PlusPaclitaxel

The patient is a 62 year-old male with relapsed diffuse large B-celllymphoma (DLBCL). After 6 courses of R-CHOP chemoimmunotherapy, he nowpresents with extensive lymph node spread in the mediastinum, axillary,and inguinal lymph nodes. He is given IMMU-114 plus paclitaxel on a21-day cycle. The anti-HLA-DR mAb is administered at a dose of 12 mg/kgon days 1 and 7 and paclitaxel is administered at 175 mg/m² on days 1, 7and 14 of the cycle. After 3 cycles, the patient is evaluated by CTimaging, and his total tumor bulk is measured and shows a decrease of35% (partial response), which appears to be maintained over the next 3months. Side effects are only thrombocytopenia and grade 1 nausea andvomiting after therapy, which resolve within 2 weeks. No pretherapy forreducing infusion reactions is given.

Example 10 Frontline Therapy of Follicular Lymphoma Using IMMU-140 andRucaparib

The patient is a 41-year-old woman presenting with low-grade follicularlymphoma, with measurable bilateral cervical and axillary lymph nodes(2-3 cm each), mediastinal mass of 4 cm diameter, and an enlargedspleen. She is given IMMU-140 in combination with rucaparib, with ADCadministered at 10 mg/kg on day 1 and rucaparib at 12 mg/m² on days 1, 8and 15. After 4 cycles, her tumor measurements by CT show a reduction of80%. She is then given 2 additional courses of therapy, and CTmeasurements indicate that a complete response is achieved. This isconfirmed by FDG-PET imaging.

Example 11 Production and Use of Pro-2-Pyrrolinodoxorubicin (P2PDox)

Pro-2-pyrrolinodoxorubicin (P2PDox) was synthesized and conjugated toantibodies as described in U.S. Pat. No. 8,877,202, the Figures andExamples section of which are incorporated herein by reference.Exemplaray P2PDox ADCs are disclosed in Table 5 below.

TABLE 5 Exemplary P2PDox-Antibody Conjugates % HPLC Protein P2PDo FreeConjugate Lot recover x/IgG Aggr. drug 1 hIMMU-31- II22-138 75.0% 7.391.9% 0.26% P2PDox 2 hA20-P2PDox II22-135 85.7% 6.79 <2% <0.1% 3hLL1-P2PDox II22-145 88.6% 7.10 2.8%  0.2% 4 hRS7-P2PDox II22-142 80.1%7.17 1.8% 0.12% 5 hMN15- II22-180 74.9% 6.87 1.1% 0.46% P2PDox 6 hMN-14-II22-183 80.2% 6.78 2.1% 0.53% P2PDox

Conjugates were also prepared for hPAM4-P2PDox, hLL2-P2PDox andRFB4-P2PDox, with similar protein recovery and purity (not shown).

In Vitro Cell-Binding Studies—

Retention of antibody binding was confirmed by cell binding assayscomparing binding of the conjugate to unconjugated antibody (Chari,2008, Acc Chem Res 41:98-107). The potency of the conjugate was testedin a 4-day MTS assay using appropriate target cells. The hRS7-P2PDoxconjugate exhibited IC₅₀ values of 0.35-1.09 nM in gastric (NCI-N87),pancreatic (Capan-1), and breast (MDA-MB-468) human cancer cell lines,with free drug exhibiting 0.02-0.07 nM potency in the same cell lines.

Serum Stability—

Serum stability of prototypical P2PDox conjugate, hRS7-P2PDox, wasdetermined by incubating in human serum at a concentration of 0.2 mg/mLat 37° C. The incubate was analyzed by HPLC using butyl hydrophobicinteraction chromatography (HIC) column in which there was goodretention time separation between the peak due to free drug and that dueto conjugate or higher molecular weight species. This analysis showedthat there was no release of free drug from the conjugate, suggestinghigh serum stability of the conjugate. When the same experiment wasrepeated with hRS7-doxorubicin conjugate, containing the same cleavablelinker as hRS7-P2PDox, and where the free drug was independentlyverified to be released with a half-life of 96 h, clear formation offree drug peak, namely doxorubicin peak, was seen on HIC HPLC.

Surprisingly, it was determined that the P2PDox conjugate was heldtightly to the antibody because it cross-linked the peptide chains ofthe antibody together. The cross-linking stabilizes the attachment ofthe drug to the antibody so that the drug is only releasedintracellularly after the antibody is metabolized. The cross-linkingassists in minimizing toxicity, for example cardiotoxicity, that wouldresult from release of free drug in circulation. Previous use of 2-PDoxpeptide conjugates failed because the drug cross-linked the peptide toother proteins or peptides in vivo. With the present conjugates, theP2PDox is attached to interchain disulfide thiol groups while in theprodrug form. The prodrug protection is rapidly removed in vivo soonafter injection and the resulting 2-PDox portion of the conjugatecross-links the peptide chains of the antibody, forming intramolecularcross-linking within the antibody molecule. This both stabilizes the ADCand prevents cross-linking to other molecules in circulation.

Example 12 In Vivo Studies with P2PDox-Conjugated Antibodies

General—

Tumor size was determined by caliper measurements of length (L) andwidth (W) with tumor volume calculated as (L×W²)/2. Tumors were measuredand mice weighed twice a week. Mice were euthanized if their tumorsreached >1 cm³ in size, lost greater than 15% of their starting bodyweight, or otherwise became moribund. Statistical analysis for the tumorgrowth data was based on area under the curve (AUC) and survival time.Profiles of individual tumor growth were obtained through linear curvemodeling. An f-test was employed to determine equality of variancebetween groups prior to statistical analysis of growth curves. Atwo-tailed t-test was used to assess statistical significance betweenall the various treatment groups and non-specific controls. For thesaline control analysis a one-tailed t-test was used to assesssignificance. Survival studies were analyzed using Kaplan-Meier plots(log-rank analysis), using the Prism GraphPad Software (v4.03) softwarepackage (Advanced Graphics Software, Inc.; Encinitas, Calif.). All dosesin preclinical experiments are expressed in antibody amounts. In termsof drug, 100 μg of antibody (5 mg/kg) in a 20-g mouse, for example,carries 1.4 μg-2.8 μG (0.14-0.17 mg/kg) of P2PDox equivalent dose whenusing an ADC with 3-6 drugs/IgG.

A single i.v. dose of ≥300 μg [˜10 μg of P2PDox] of the conjugate waslethal, but 4 doses of 45 μg given in 2 weeks were tolerated by allanimals. Using this dosing regimen, we examined the therapeutic effectof hRS7-P2PDox in 2 human tumor xenograft models, Capan-1 (pancreaticcancer) and NCI-N87 (gastric cancer). Therapy began 7 days after tumortransplantation in nude mice. In the established, 7-day-old, Capan-1model, 100% of established tumors quickly regressed, with no evidence ofre-growth (not shown). This result was reproduced in a repeat experiment(not shown). Similar findings were made in the established NCI-N87 model(not shown), where a 2^(nd) course of therapy, administered after day70, was safely tolerated and led to further regressions of residualtumor (not shown). The internalizing hRS7-SN-38 conjugate, targetingTrop-2, provided better therapeutic responses than a conjugate of apoorly internalizing anti-CEACAM-5 antibody, hMN-14 (not shown). Anon-targeted anti-CD20 ADC, hA20-P2PDox, was ineffective, indicatingselective therapeutic efficacy (not shown). Data from a breast cancerxenograft (MDA-MB-468) and a second pancreatic cancer xenograft (notshown) reiterate the same trend of the conjugate's specific andsignificant antitumor effects.

PK and Toxicity of hRS7-P2PDox with Substitutions of 6.8 or 3.7Drug/IgG—

Antibody-drug conjugates (ADCs) carrying as much as 8 ultratoxicdrugs/MAb are known to clear faster than unmodified MAb and to increaseoff-target toxicity, a finding that has led to the current trends to usedrug substitutions of ≤4 (Hamblett et al., 2004, Clin Cancer Res10:7063-70). Conjugates were prepared and evaluated with mean drug/MAbsubstitution ratios (MSRs) of ˜6:1 and ˜3:1. Groups of normal mice (n=5)were administered, i.v., single doses of unmodified hRS7 or hRS7-P2PDoxwith drug substitution of 6.8 or 3.7 (same protein dose), and serumsamples were collected at 30 min, 4 h, 24 h, 72 h, and 168 hpost-injection. These were analyzed by ELISA for antibody concentration(not shown). There were no significant differences in serumconcentrations at various times, indicating that these clearedsimilarly. The PK parameters (Cmax, AUC, etc.) were similar. Conjugateswith either higher or lower drug substitution had similar tolerabilityin nude mice, when the administered at the same dose of conjugated drug.

Therapeutic Efficacy at Minimum Effective Dose (MED)—

Anti-TROP-2 antibody conjugate, hRS7-P2PDox, was evaluated in nude micebearing NCI-N87 human gastric cancer xenografts by administering asingle bolus protein dose of 9 mg/kg, 6.75 mg/kg, 4.5 mg/kg, 2.25 mg/kg,or 1 mg/kg. The therapy was started when the mean tumor volume (mTV) was0.256 cm³. On day 21, mTV in the saline control group (non-treatmentgroup) was 0.801±0.181 cm³ which was significantly larger than that inmice treated with 9, 6.75, 4.5, or 2.25 mg/kg dose with mTV of0.211±0.042 cm³, 0.239±0.0.054 cm³, 0.264±0.087 cm³, and 0.567±0.179cm³, respectively (P<0.0047, one tailed t-test). From these, the minimumeffective dose was judged to be 2.25 mg/kg, while 9 mg/kg representedMTD.

MTD of Antibody-P2PDox—

An MTD study comparing 2-PDox and P2PDox conjugates of prototypeantibody, hLL1, in mice demonstrated that the P2PDox conjugate was muchmore potent (not shown). The MTD of a single i.v. injection was between100 and 300 μg. The MTD of multiple injections, at a schedule of everyfour days for a total of four injections (q4dx4) was then determined,using protein doses between 25 μg to 150 μg per injection. At thesedoses, a cumulative dose of between 100 and 600 μg was given to theanimals. Table 6 below summarizes the various groups.

TABLE 6 Dosage and Schedule for MTD of antibody-P2PDox 12 Female AthymicNude Mice Group N Treatment Total Amount 1 3 25 μg i.v. q4dx4 100 μg 2 350 μg i.v. q4dx4 200 μg 3 3 100 μg i.v. q4dx4 400 μg 4 3 150 μg i.v.q4dx4 600 μg

Only those mice treated with 25 μg P2PDox-ADC continue to show no signsof toxicity (not shown). This is a cumulative dose of 100 μg which wasalso the dose tolerated when administered as a single injection (notshown). Therefore, the MTD for multiple injections of a P2PDox-ADC inmice is 25 μg q4dx4 from this experiment. A more detailed analysis ofdata and repetition of the experiment established the MTD forfractionated dosing to be 45 μg of protein dose of the conjugate,administered every 4 days for 2 weeks (45 q4dx4 schedule).

Binding Studies—

No significant difference in binding of the antibody moiety to NCI-N87gastric carcinoma cells was observed between unconjugated hRS7 andP2PDox-hRS7 conjugated to 6 molecules of P2PDox per antibody (notshown). The lack of effect of conjugation on antibody binding to targetantigen was confirmed for P2PDox-hMN-15 (anti-CEACAM-6), P2PDox-hLL2(anti-CD22) and P2PDox-hMN-24 (anti-CEACAM-5) conjugates. It isconcluded that conjugation of P2PDox to antibodies does not affectantibody-antigen binding activity.

Cytotoxicity Studies—

The cytotoxicity of P2PDox-mAb conjugates to target cells was examined.hRS7-P2PDox and hMN-15-P2PDox were cytotoxic to MDA-MB-468, AG S,NCI-N87 and Capan-1 solid tumor cell lines (not shown). hMN-14-P2PDoxwas cytotoxic to Capan-1, BxPC-3 and AsPC-1 human pancreatic tumor linesand AGS, NCI-N87 and LS147T human gastric and colonic tumor lines (notshown). hLL2-P2PDOx was cytotoxic to Daudi, Raji, Ramos and JVM-3hematopoietic tumor lines (not shown). IC₅₀ values for the conjugateswere in the nanomolar concentration range (not shown).

Example 13 Further in Vivo Studies

Further in vivo efficacy studies were performed in nude mice implantedwith NCI-N87 human gastric cancer xenografts (not shown). One treatmentcycle with 4×45 ∝g of hRS7-P2PDox rapidly regressed all tumors (notshown). A second treatment cycle was initiated about 2 months after theend of the first cycle, resulting in complete regression of all but oneof the hRS7-P2PDox treated animals. The hA20, hLL1 and hMN-14 conjugateshad little effect on tumor progression (not shown). Administration ofP2PDox-hMN-15 resulted in a delayed regression of gastric cancer, whichwas less effective than the hRS7 conjugate.

The effect of varying dosage schedule on anti-tumor efficacy wasexamined (not shown). The experiment began 9 days after tumorimplantation when mean tumor volume for all groups was 0.383 cm³, andended on day 93 (84 days after initiation of therapy). In this study, asingle dose of 180 μg, two weekly doses of 90 μs, and q4dx4 of 45 μg allresulted in significantly enhanced survival (not shown). For the salinecontrol, 0 of 9 mice survived (not shown). For mice receiving 45 μgq4dx4 of hRS7-P2PDox, 8 of 9 mice were alive at day 94 (not shown). Formice receiving 90 μg weekly×2 of hRS7-P2PDox, 9 of 9 mice were alive atday 94 (not shown). For mice receiving a single dose of 180 μg ofhRS7-P2PDox, 8 of 9 mice were alive at day 94 (not shown). At the samedosage schedule, the control hA20 conjugate had no effect on survival(not shown). A toxicity study showed that the three dosage schedules ofhRS7-P2PDox resulted in similarly low levels of toxicity (not shown).

The hRS7-P2PDox conjugate was also effective in Capan-1 pancreaticcancer (not shown) and was more effective at inhibiting tumor growththan a hRS7-SN-38 conjugate (not shown). The hPAM4-P2PDox conjugate wasalso more effective at inhibiting growth of Capan-1 human pancreaticcancer than an hPAM4-SN-38 conjugate (not shown). At 63 days afterCapan-1 tumor injection (with therapy starting at 1 dayspost-innoculation), 0 of 10 mice were alive in the saline control, 10 of10 mice were alive in mice treated twice weekly×2 weeks with 45 μg ofhPAM4-P2PDox, 2 of 10 mice were alive in mice treated twice weekly×2weeks with 45 μg of hA20-P2PDox, 0 of 10 mice were alive in mice treatedtwice weekly×4 weeks with 250 μg of hPAM4-SN-38, and 0 of 10 mice werealive in mice treated twice weekly×4 weeks with 250 μg of h20-SN-38.

hRS7-P2PDox was substantially more effective than hRS7-SN-38 atinhibiting growth of PxPC-3 pancreatic cancer (not shown) and wasslightly more effective than hRS7-SN-38 at inhibiting growth ofMDA-MB-468 breast cancer (not shown).

The effect of different single doses of hRS7-P2PDox on growth of NCI-N87gastric carcinoma xenografts was examined. Using a single dose, themaximum effect on tumor growth was observed at 90 μg or higher (notshown). A single dose of 45 μg was the minimum required to see asignificant survival benefit compared to saline control (not shown).

The ADCC activity of various hRS7-ADC conjugates was determined incomparison to hRS7 IgG (not shown). PBMCs were purified from bloodpurchased from the Blood Center of New Jersey. A Trop-2-positive humanpancreatic adenocarcinoma cell line (BxPC-3) was used as the target cellline with an effector to target ratio of 100:1. ADCC mediated by hRS7IgG was compared to hRS7-Pro-2-PDox, hRS7-CL2A-SN-38, and the reducedand capped hRS7-NEM. All were used at 33.3 nM. Overall activity was low,but significant (not shown). There was 8.5% specific lysis for the hRS7IgG which was not significantly different from hRS7-Pro-2-PDox. Bothwere significantly better than hLL2 control and hRS7-NEM and hRS7-SN-38(P<0.02, two-tailed t-test). There was no difference between hRS7-NEMand hRS7-SN-38.

Example 14 Immunoconjugate Storage

The ADC conjugates are were purified and buffer-exchanged with2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, and furtherformulated with trehalose (25 mM final concentration) and polysorbate 80(0.01% v/v final concentration), with the final buffer concentrationbecoming 22.25 mM as a result of excipient addition. The formulatedconjugates are lyophilized and stored in sealed vials, with storage at2° C.-8° C. The lyophilized ADCs are stable under the storage conditionsand maintain their physiological activities.

Example 15 Summary of Results with IMMU-140 (SN-38 Conjugated IMMU-114)in Hematopoietic Cancer Xenografts

Relapsed AML (acute myeloid leukemia), ALL (acute lymphatic leukemia),and MM (multiple myeloma) continue to be a therapy challenge. IMMU-114(hL243) is a humanized anti-HLA-DR IgG4 monoclonal antibody engineeredto lack effector-cell functions, but retains HLA-DR binding and a broadrange of antitumor effects in diverse hematological neoplasms (Stein etal., Blood. 2010; 115:5180-90). When given subcutaneously, it hasencouraging efficacy in an initial Phase I clinical trial in relapsed orrefractory NHL (non-Hodgkin's lymphoma) and CLL (chronic lymphaticleukemia), with a good safety profile (ClinicalTrials.gov, NCT01728207).

In vitro, AML has proven to be resistant to the antitumor effects ofIMMU-114, despite high expression levels of HLA-DR. Likewise, in severaldifferent human ALL and MM cell lines, IMMU-114 has demonstrated a rangeof antitumor effects from a low of 9% to a high of 69%. In an effort toimprove the antitumor activity of IMMU-114, an antibody-drug conjugate(ADC) was made in which IMMU-114 was conjugated with the activemetabolite of irinotecan, SN-38, with the new conjugate designatedIMMU-140. Another ADC utilizing SN-38 (sacituzumab govitecan) beingstudied in solid tumors has been well tolerated, with clinicallysignificant objective responses in patients given multiple cyclesover >6 months, with manageable neutropenia being the major toxicity.Thus, our goal was to determine if SN-38, a drug not commonly used inhematopoietic cancers, would prove to be an effective and safetherapeutic when targeted with IMMU-140. In this current work, the invivo activity of IMMU-140 versus parental IMMU-114 was examined in humanAML, ALL, and MM xenografts.

Conjugation of SN-38 to hL243 IgG4 resulted in a drug-to-antibody-ratiorange of 6.1 to 6.6. For AML and MM disease models, NSG/SCID and C.B.-17SCID mice received 2 Gy irradiation 24 h prior to an i.v. injection ofMOLM-14 (2×10⁶) or CAG cells (1×10⁷), respectively. ALL was establishedin C.B-17 SCID mice injected with MN-60 cells (1×10⁷). All therapiesbegan 5 days post-tumor-cell injection. Test agents, including anon-targeting anti-CEA-SN-38 ADC, were administered as 500-μg injectionstwice-weekly for 4 wks. Animals were sacrificed at disease progression,characterized by the onset of hind-limb paralysis or loss of more than15% body weight.

As discussed in the following Examples, in experimental AML, salinecontrol and IMMU-114 treated mice succumbed to disease progressionquickly, with a median survival time (MST) of only 14 and 15 days,respectively. Conversely, mice treated with IMMU-140 had a greater than1.5-fold increase in survival (MST=37 d, P=0.0031). Further, IMMU-140therapy provided a significant survival benefit when compared toanti-CEA-SN-38 control (MST=21 d, P=0.0031). In mice bearing ALLxenografts, IMMU-114 provided a >60% improvement in survival compared tosaline control (MST=37 d vs. 22.5 d, respectively; P<0.0001), whereasIMMU-140 increased this by another 80% (MST=66.5 d) which wassignificantly better than all other treatments, including IMMU-114(P<0.0001). Finally, MM mice had a greater than 123-d MST when treatedwith IMMU-140 compared to 32 d for saline control (P<0.0001). Thissurvival benefit also was significantly higher than for mice treatedwith bortezomib (0.89 mg/kg) or control ADC (MST=32.5 d for both;P<0.0001). While not significant, IMMU-140 does provide a >30%improvement in survival when compared to IMMU-114 therapy (MST=94.5 d,P=0.1313). In all three experiments, therapy with IMMU-140 was welltolerated, as evidenced by no significant loss in body weight.

Based on these results, therapy with the IMMU-140 anti-HLA-DR ADC provedto be superior to IMMU-114 (which is active clinically in NHL and CLL)in both AML and ALL xenografts and beneficial in MM. Most importantly,in IMMU-114-refractive AML, IMMU-140 demonstrated a significantantitumor effect without any undue toxicity. The data show that this newADC is of use in these intractable malignancies.

Example 16 Efficacy of IMMU-140 in Experimental Acute Myeloid Leukemia(AML)

In a series of three different in vivo experiments, the efficacy of ananti-HLA-DR monoclonal antibody, (IMMU-114; hL243-γ4P), was compared toan ADC (IMMU-140) made with IMMU-114 conjugated to SN-38, in micebearing experimental human acute myeloid leukemia (AML), acute lymphaticleukemia (ALL), and multiple myeloma (MM).

Experimental Design.

NSG/SCID mice were irradiated (2 Gy) followed 24 h later byadministration of MOLM-14 cells (2×10⁶ cells i.v.). At the time of cellinjection, the mice were randomized into six treatment groups of 5 miceeach. Five days later mice began therapy. IMMU-114 was administered as500 μg s.c. injections twice weekly for four weeks. Likewise, IMMU-140(drug-antibody ratio DAR=6.7) was administered as 500 μg or 250 μg i.p.injections twice weekly for four weeks. Control animals received anon-tumor-targeting anti-CEACAM5 ADC (hMN14-SN-38; DAR=6.1). Table 7summarizes the various treatment groups.

TABLE 7 IMMU-114 versus IMMU-140 Efficacy in a Human AML Disease Model(MOLM-14). Schedule (Begin 5 days post-cell Group (N) Treatmentinjection) 1 5 Saline Twice weekly × 4 wks (100 μL i.p.) 2 5 IMMU-140Twice weekly × 4 wks (500 μg i.p.) 3 5 IMMU-140 Twice weekly × 4 wks(250 μg i.p.) 4 5 hMN14-SN-38 Twice weekly × 4 wks (500 μg i.p.) 5 5hMN14-SN-38 Twice weekly × 4 wks (250 μg i.p.) 6 5 IMMU-114 Twice weekly× 4 wks (500 μg s.c.)

Mice were monitored daily and were sacrificed at disease progression,characterized by the onset of hind-limb paralysis or loss of more than15% body weight. Survival was analyzed using Kaplan-Meier plots(log-rank analysis), using the Prism GraphPad Software (v6.05) package(Advanced Graphics Software, Inc.; Encinitas, Calif.).

Results

Saline control and IMMU-114 treated mice succumbed to diseaseprogression quickly, with a median survival time (MST) of only 14 and 15days, respectively (FIG. 9). Conversely, mice treated with IMMU-140 hada greater than 1.5-fold increase in survival (MST=37 d, P=0.0031) (FIG.9). Further, IMMU-140 therapy provided a significant survival benefitwhen compared to anti-CEA-SN-38 control (MST=21 d, P=0.0031) (FIG. 9). Adose-reduction to 250 μg IMMU-140 still provided a greater than 80%improvement in survival compared to saline and control ADC at the samedose (P=0.0031) (FIG. 9). Therapy with IMMU-140 was well tolerated, asevidenced by no significant loss in body weight (not shown).

Example 17 Efficacy of IMMU-140 in Experimental Acute Lymphatic Leukemia(ALL)

Experimental Design.

In this experiment, C.B.-17 SCID mice were injected with the MN-60 humanALL cell line (1×10⁷ cells). At the time of cell injection, the micewere randomized into five treatment groups of 10 mice each. Five dayslater mice began therapy. IMMU-114 was administered as 500 μg s.c.injections twice weekly for four weeks. Likewise, IMMU-140 (DAR=6.2) wasadministered as 500 μg i.p. injections twice weekly for four weeks.Control animals received non-targeting hMN-14 IgG or hMN14-SN-38(DAR=6.1). Treatment groups are summarized in Table 8 below.

TABLE 8 Treatment of ALL with IMMU-140 vs. IMMU-114 IMMU-114 andIMMU-140 Efficacy in a Human ALL Disease Model (MN-60). Schedule (Begin5 days post-cell Group N Treatment injection) 1 10 Saline Twice weekly ×4 wks (100 μL i.p.) 2 10 IMMU-114 Twice weekly × 4 wks (500 μg s.c.) 310 IMMU-140 Twice weekly × 4 wks (500 μg i.p.) 4 10 hMN-14 Twice weekly× 4 wks (500 μg s.c.) 5 10 hMN14-SN-38 Twice weekly × 4 wks (500 μgi.p.)

Mice were monitored daily and were sacrificed at disease progression,characterized by the onset of hind-limb paralysis or loss of more than15% body weight. Survival was analyzed using Kaplan-Meier plots(log-rank analysis), using the Prism GraphPad Software (v6.05) package(Advanced Graphics Software, Inc.; Encinitas, Calif.).

Results

As shown in FIG. 10, IMMU-114 provided a >60% improvement in survivalcompared to saline control (MST=37 d vs. 22.5 d, respectively;P<0.0001), whereas IMMU-140 increased this by another 80% (MST=66.5 d)which was significantly better than all other treatments, includingIMMU-114 (P<0.0001). When the study ended on day 105, three mice in theIMMU-140 group (30%) were still alive. Upon necropsy, there was novisible evidence of disease in these mice (i.e., no internal tumorsfound). Therapy with IMMU-140 was well tolerated, as evidenced by nosignificant loss in body weight.

Example 18 Efficacy in Experimental Multiple Myeloma (MM)

Experimental Design

C.B-17 SCID mice were irradiated with 2 Gy one day before being injectedi.v. with CAG cells (1×10⁷ cells). At the time of cell injection, themice were randomized into eight treatment groups of 10 mice each. Fivedays later mice began therapy as outlined in the table below. For theADCs, IMMU-140 had a DAR=6.1 and control hMN14-SN-38 had a DAR=6.6.Clinically, bortezomib is administered to MM patients at 1.3 mg/m² twicea week for two weeks followed by one week rest before repeating. Aclinical dose of 1.3 mg/m² is equivalent to 0.43 mg/kg to mice. For thisstudy, mice were administered bortezomib (15 μg; 0.89 mg/kg) weekly forfour weeks only. Treatment groups are summarized Table 9.

Mice were monitored daily and were sacrificed at disease progression,characterized by the onset of hind-limb paralysis or loss of more than15% body weight. Survival was analyzed using Kaplan-Meier plots(log-rank analysis), using the Prism GraphPad Software (v6.05) package(Advanced Graphics Software, Inc.; Encinitas, Calif.).

TABLE 9 Efficacy of IMMU-114 vs. IMMU-140 in multiple myeloma xenograftsIMMU-114 and IMMU-140 Efficacy in a Human MM Disease Model (CAG).Schedule (Begin 5 days post-cell Group N Treatment injection) 1 10Saline Twice weekly × 4 wks (100 μL i.p.) 2 10 IMMU-114 Twice weekly × 4wks (500 μg i.p.) 3 10 IMMU-140 Twice weekly × 4 wks (500 μg i.p.) 4 10Bortezomib Weekly × wks (15 μg; ~1 mg/kg i.v.) 5 10 IMMU-114 Twiceweekly × 4 wks (500 μg i.p.) + + Weekly × 4 wks Bortezomib (15 μg; ~1mg/kg i.v.) 6 10 IMMU-140 Twice weekly × 4 wks (500 μg i.p.) + + Weekly× 4 wks Bortezomib (15 μg; ~1 mg/kg i.v.) 7 10 hMN-14 IgG Twice weekly ×4 wks (500 μg i.p.) + + Weekly × 4 wks Bortezomib (15 μg; ~1 mg/kg i.v.)8 10 hMN14-SN-38 Twice weekly × 4 wks (500 μg i.p.) + + Weekly × 4 wksBortezomib (15 μg; ~1 mg/kg i.v.)

Results

As shown in the graph below, there is a greater than 126-d MST in micetreated with IMMU-140 compared to 32 d for saline control (P<0.0001).This survival benefit also is significantly higher than for mice treatedwith bortezomib (0.89 mg/kg) or control ADC (MST=32.5 d for both;P<0.0001). While not significant, IMMU-140 does provide a >30%improvement in survival when compared to IMMU-114 therapy (MST=94.5 d,P=0.2162). The addition of bortezomib treatment to either IMMU-140 orIMMU-114 therapy has not provided a significant survival benefit abovethat observed with monotherapy of each. Therapy with IMMU-140 was welltolerated, as evidenced by no significant loss in body weight.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usage andconditions without undue experimentation. All patents, patentapplications and publications cited herein are incorporated byreference.

What is claimed is:
 1. A method of treating a hematopoietic cancer,comprising: a) administering to a subject with a hematopoietic cancer anIMMU-140 antibody-drug conjugate (ADC) that binds to HLA-DR; and b)administering to the subject a PI3K (phosphoinositide 3-kinase)inhibitor.
 2. The method of claim 1, wherein the IMMU-140 ADC comprisesthe heavy chain complementarity determining region (CDR) sequences CDR1(NYGMN, SEQ ID NO: 1), CDR2 (WINTYTREPTYADDFKG, SEQ ID NO:2), and CDR3(DITAVVPTGFDY, SEQ ID NO:3) and the light chain CDR sequences CDR1(RASENIYSNLA, SEQ ID NO:4), CDR2 (AASNLAD, SEQ ID NO:5), and CDR3(QHFWTTPWA, SEQ ID NO:6).
 3. The method of claim 1, wherein the PI3Kinhibitor is selected from the group consisting of idelalisib,Wortmannin, demethoxyviridin, perifosine, PX-866, IPI-145 (duvelisib),BAY 80-6946, BEZ235, RP6530, TGR1202, SF1126, INK1117, GDC-0941, BKM120,XL147, XL765, Palomid 529, GSK1059615, ZSTK474, PWT33597, IC87114,TG100-115, CAL263, PI-103, GNE477, CUDC-907, AEZS-136 and LY294002. 4.The method of claim 1, wherein the PI3K inhibitor is idelalisib.
 5. Themethod of claim 1, further comprising administering to the subject aPARP inhibitor selected from the group consisting of olaparib,talazoparib (BMN-673), rucaparib, veliparib, CEP 9722, MK 4827, BGB-290,ABT-888, AG014699, BSI-201, CEP-8983 and 3-aminobenzamide.
 6. The methodof claim 5, wherein the PARP inhibitor is olaparib.
 7. The method ofclaim 1, further comprising administering to the subject a microtubuleinhibitor selected from the group consisting of a vinca alkaloid, ataxane, a maytansinoid, an auristatin, vincristine, vinblastine,paclitaxel, mertansine, demecolcine, nocodazole, epothilone, docetaxel,disodermolide, colchicine, combrestatin, podophyllotoxin, CI-980,phenylahistins, steganacins, curacins, 2-methoxy estradiol, E7010,methoxy benzenesuflonamides, vinorelbine, vinflunine, vindesine,dolastatins, spongistatin, rhizoxin, tasidotin, halichondrins,hemiasterlins, cryptophycin 52, MMAE and eribulin mesylate.
 8. Themethod of claim 7, wherein the microtubule inhibitor is paclitaxel oreribulin mesylate.
 9. The method of claim 1, further comprisingadministering to the subject a Bruton's tyrosine kinase (BTK) inhibitor.10. The method of claim 9, wherein the BTK inhibitor is selected fromthe group consisting of PCI-45292, CC-292 (AVL-292), ONO-4059, GDC-0834,LFM-A13 and RN486.
 11. The method of claim 1, further comprisingadministering to the subject one or more additional therapeuticmodalities selected from the group consisting of unconjugatedantibodies, immunoconjugates, radiolabeled antibodies, drugs, toxins,gene therapy, chemotherapy, therapeutic peptides, cytokine therapy,localized radiation therapy, surgery and interference RNA therapy. 12.The method of claim 11, wherein the therapeutic modality comprisestreatment with an agent selected from the group consisting of5-fluorouracil, afatinib, aplidin, azaribine, anastrozole,anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin,bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin,camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine,celecoxib, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan(CPT-11), SN-38, carboplatin, cladribine, camptothecans,cyclophosphamide, crizotinib, cytarabine, dacarbazine, dasatinib,dinaciclib, docetaxel, dactinomycin, daunorubicin, doxorubicin,cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicinglucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib,entinostat, estrogen receptor binding agents, etoposide, etoposideglucuronide, etoposide phosphate, exemestane, fingolimod, flavopiridol,floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine,flutamide, farnesyl-protein transferase inhibitors, fostamatinib,ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea,idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase, lapatinib,lenolidamide, leucovorin, LFM-A13, lomustine, mechlorethamine,melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone,mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib,nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765,pentostatin, PSI-341, raloxifene, semustine, sorafenib, streptozocin,SU11248, sunitinib, tamoxifen, temazolomide, transplatinum, thalidomide,thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vatalanib,vinorelbine, vinblastine, vincristine, vinca alkaloids and ZD1839. 13.The method of claim 1, wherein the cancer is selected from the groupconsisting of B-cell lymphoma, B-cell leukemia, myeloid leukemia, andmultiple myeloma.
 14. The method of claim 13, wherein the B-cellleukemia or B-cell lymphoma is selected from the group consisting ofindolent forms of B-cell lymphoma, aggressive forms of B-cell lymphoma,chronic lymphocytic leukemia, acute lymphocytic leukemia, hairy cellleukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, Burkitt'slymphoma, follicular lymphoma, diffuse B-cell lymphoma, mantle celllymphoma and multiple myeloma.